# Photodynamic Therapy Using Pulsed-Laser Irradiation of Gold Nanoparticles for the Treatment of Cancers with High Basal Oxidative Stress

Léa Tourneur, Syrine Gueffrache, Khanh-Van Do, Justine Paris, Yoann Lalatonne, Erwann Guénin, Diaddin Hamdan, Charlotte Comte, Alexandra Marsocci, Benoît Caron, et al.

► To cite this version:

Léa Tourneur, Syrine Gueffrache, Khanh-Van Do, Justine Paris, Yoann Lalatonne, et al. "Photodynamic Therapy Using Pulsed-Laser Irradiation of Gold Nanoparticles for the Treatment of Cancers with High Basal Oxidative Stress." Nano Letters, 2025, 25 (36), pp.13655-13663. 10.1021/acs.nanolett.5c03580. hal-05337767

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Photodynamic Therapy using Pulsed-Laser Irradiation of Gold Nanoparticles for the Treatment of Cancers with High Basal Oxidative Stress

Léa Tourneur<sup>a,b&</sup>, Syrine Gueffrache<sup>c&</sup>, Khanh-van Do<sup>c</sup>, Justine Paris<sup>d</sup>, Yoann Lalatonne<sup>e,f</sup>, Erwann Guénin<sup>g</sup>, Diaddin Hamdan<sup>h</sup>, Charlotte Comte<sup>b</sup>, Alexandra Marsocci<sup>b</sup>, Benoît Caron<sup>i</sup>, Emmanuel Van Glabeke<sup>j</sup>, Sylvie Marguet<sup>k</sup>, Christophe Leboeuf<sup>a</sup>, Bruno Palpant<sup>c#</sup>, Guilhem Bousquet<sup>b,l*#</sup>

<sup>a</sup>Université Paris Cité, F-75006, Paris, France

<sup>b</sup>Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France

<sup>c</sup>Université Paris-Saclay, CNRS, ENS Paris-Saclay, CentraleSupélec, LuMIn, Gif <sup>s</sup>/Yvette, France

<sup>d</sup>Université Paris Cité, F-75006, Paris, France

<sup>e</sup>LVTS, INSERM, U1148, F-93000, Bobigny, F-93000, France

<sup>f</sup>AP-HP Hôpital Avicenne, Service de médecine nucléaire, F-93100, Bobigny, France

<sup>g</sup>Sorbonne Université, Université de Technologie de Compiègne, Integrated Transformations of Renewable Matter Laboratory, (EA TIMR 4297 UTC-ESCOM), F-60200, Compiègne, France

<sup>h</sup>Hôpital la Porte Verte, F-78000 Versailles, France

<sup>i</sup>ALIPP6, Institut des Sciences de la Terre de Paris UMR 7193, CNRS, Sorbonne Université, F-75005 Paris, France

<sup>j</sup>Fédération d'Urologie de Seine-Saint-Denis, F-93100, Montreuil, F-93100, France

<sup>k</sup>Université Paris-Saclay, CEA, CNRS, NIMBE, 91191, Gif-sur-Yvette, France.

<sup>l</sup>AP-HP Hôpital Avicenne, Service d'oncologie médicale, F-93100, Bobigny, France

<sup>&amp;</sup>These authors are co-first authors

<sup>#</sup>These authors are co-senior authors

<sup>*</sup>Correspondence: Guilhem Bousquet, Université Sorbonne Paris Nord, SMBH rue Marcel Cachin, Bobigny, F-93000, France, Email: guilhem.bousquet@aphp.fr; Tel: 0033681845379
ABSTRACT: For over twenty years, considerable research has been carried out on the use of nanotechnologies in cancer treatment. Beyond the photothermal effect of irradiating gold nanoparticles, the photodynamic effect with reactive oxygen species (ROS) production at tissue level is poorly known. We showed that renal cancers had high basal ROS levels. We engineered different gold nanoparticles and optimized irradiation parameters. Then, we intravenously injected several patient-derived xenografts with tailored gold nanorods that we irradiated using pulsed laser. We optimized irradiation parameters to decrease toxic effects on normal tissue, and showed that the local monitoring of skin temperature was an accurate marker for predicting histological toxicity. We demonstrated a significant additional effect of irradiating gold nanoparticles, with the increased activation of autophagy pathway following irradiation, associated with the disappearance of renal cancer cells. Our study highlights the elevated ROS levels in cancers as a potential therapeutic target for ROS-mediated cytotoxicity therapies.

KEYWORDS: gold nanoparticles, photothermal therapy, photodynamic therapy, femtosecond laser, reactive oxygen species, autophagy
For more than twenty years, considerable research has been carried out on the use of nanotechnologies in the treatment of cancers, including metallic nanoparticles with still very limited clinical success.¹,² The heterogeneity of the nanoparticles engineered, the different methods used to produce them, without systematic optimization of the therapeutic parameters, could be one explanation for this relative failure.³,⁴ To target cancer cells, gold nanoparticles are attractive candidates, since specific antibodies can be conjugated to their surface.⁵,⁶,⁷ In addition, the physical properties of gold make these nanoparticles suitable for photothermal and photodynamic therapies using near-infrared laser irradiation,⁸ as well as for drug or oligonucleotide delivery.⁹,¹⁰ Recently, using multifunctional gold nanoparticles coated with anti-HER2 antibody and illuminated with femtosecond laser pulses, our research team demonstrated the feasibility of targeting breast cancer xenografts to overcome acquired resistance to trastuzumab.⁶ Irradiation with ultrashort laser pulses is more efficacious for very localised overheating than irradiation with continuous laser light, because it produces very circumscribed heat bursts in the vicinity of the nanoscale thermal sources.¹¹ This enables the use of lower laser powers,¹² so as to limit toxicity to healthy tissues surrounding the tumors, a condition required for a translational application to patients. Beyond this, the added value of femtosecond pulsed-laser irradiation of gold nanorods could be the combination of photothermal and photodynamic effects through the surface plasmon resonance. A single 40-nm long gold nanorod is able to produce reactive oxygen species (ROS) over a range of several micrometers,¹³ and an increase in the light excitation wavelength could promote a switch from photothermal to photodynamic effects, or a combination of the two.¹⁴ But these effects remain to be demonstrated in vivo at tissue level. Regarding the irradiation wavelength, the second near-infrared window is known as the transparency window of biological tissues,¹⁵ theoretically enabling a better penetration in depth. In addition, in this window the effects on the
mitochondrial respiratory chain would be more marked with the stimulation of complexes I, II and IV and could thus contribute to the larger production of ROS.

Deregulation of the cellular metabolism is a hallmark of cancer, characterized by a metabolic switch to “aerobic glycolysis”, particularly marked in hypoxic and pseudo-hypoxic tumors, where the levels of hypoxia-inducible factors HIF1α and HIF2α transcription factors are increased.¹⁶ ROS are highly reactive molecules that can damage cellular components such as DNA, proteins and lipids. In healthy tissues, ROS are produced at low levels as by-products of normal cellular metabolism and are efficiently scavenged by antioxidant systems, keeping their levels balanced. In cancer cells, ROS production is increased and the antioxidant systems are often dysfunctional, leading to oxidative stress¹⁷ (Figure 1a). This oxidative stress can promote cancer cell survival, metastatic spread, and resistance to treatments. At higher levels, ROS can induce cell death through apoptosis, autophagy, necroptosis or ferroptosis, a phenomenon known as “ROS-mediated cytotoxicity” (Figure 1b). When a tumor is treated, increases in ROS levels can also damage normal tissue, leading to toxic side effects.¹⁸ It is thus necessary to selectively increase ROS levels in cancer cells while minimizing their impact on normal cells. Gold nanoparticles are potential tools for ROS-mediated cytotoxicity, with still limited data on cancer therapy at tissue level.¹⁹ In this study, we aimed to demonstrate the benefit of the photodynamic effect of using pulsed-laser irradiation of gold nanoparticles for the treatment of cancers with high basal levels of oxidative stress.

To study the potential of ROS-mediated cytotoxicity as a cancer therapy, we set out to identify tumors with high basal oxidative stress. We first compared the basal levels of ROS products and the different cell-death pathways in a panel of patient-derived xenografts reflecting human cancer heterogeneity.²⁰,²¹ We chose patient-derived xenografts from clear-cell renal cell carcinomas (ccRCCs), melanomas, and triple negative breast cancers (TNBC), all of which are severe cancers that challenge daily practice at the metastatic stage.
ROS products included 4-hydroxynonenal (4-HNE) which is a marker of lipid peroxidation, 3-nitrotyrosine which is the main product of tyrosine oxidation, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) which is one of the major products of DNA oxidation. Using immunostainings, we evidenced the presence of all these ROS products in different tumor xenografts without any specific distribution (Figure 2a for renal cancer, data not shown for the two other cancer types). In addition, we could not perform any easy quantification. In contrast, using ELISA, we showed that the ccRCC and TNBC patient-derived xenograft models had significant higher levels of ROS products, including 4-HNE and nitrotyrosine, as compared to melanoma xenografts (Figure 2a). This was associated with a significant, gradient increase in necrotic areas from ccRCCs to melanomas (*P* < 0.05) (Figure 2b), spontaneous necrosis being an indirect marker of hypoxia in the tumor.²² We then assessed different cell-death pathways in these three cancer types. Autophagy markers were significantly more expressed in ccRCCs than in breast cancer and melanoma xenografts, whether for mRNA levels for *LC3*, *BECN1*, and *ATG5*, or for protein for LC3A/B (Figure 2c). Concerning apoptosis, we tested the anti-apoptotic *BCL2* marker, for which ubiquitination, and thus inactivation, is increased by ROS. Its mRNA expression was significantly lower for ccRCCs and melanoma than for breast cancer xenografts. In contrast, the two pro-apoptotic markers, *BAX* and *BID*, were significantly overexpressed in ccRCCs and melanomas (Figure 2d). As expected, decreased *BCL2* mRNA expression and increased *BAX* and *BID* mRNAs expression were correlated with CASPASE-3 protein cleavage in melanoma. However, strikingly, no CASPASE-3 protein cleavage was observed in ccRCCs, suggesting resistance to apoptosis in these tumors (Figure 2d). To assess ferroptosis, we quantified ferrous iron Fe²⁺ and found a significantly higher level in ccRCC than in TNBC and melanoma xenografts (*P* < 0.05). Moreover, mRNA expression of the glutathione peroxidase 4 (*GPX4*), which is an inhibitor of lipid peroxidation and consequently ferroptosis, was significantly lower in ccRCC xenografts (*P* < 0.05) (Figure 2e).
ROS are at the cornerstone of several cell-death pathways, with controlled production
in normal cells to avoid oxidative stress. In cancer cells, increased but nevertheless controlled
ROS production contributes to carcinogenesis without inducing significant cell death. ROS-
mediated therapies are being developed to induce an imbalanced ROS production and trigger
cancer cell death.²³,²⁴ Using patient-derived xenografts, which fairly well reflect the
heterogeneity of human cancers,²⁵,²⁶,²⁷ we conducted an original comparative study between
three tumor types. Compared to melanomas, we showed that ccRCCs and triple-negative breast
cancers presented the highest basal levels of oxidative stress. Conversely, at basal level,
ccRCCs had small areas of spontaneous necrosis, which is an indirect marker of hypoxia,²² as
well as the highest levels of autophagy and ferroptosis markers. A possible explanation lies in
the unique genetic and metabolic profile of ccRCCs, which are pseudohypoxic and hyper-
vascularised tumors:²⁸ the almost constant loss of Von Hippel-Lindau (VHL) expression²⁹ leads
to hypoxia-inducible factor HIF1α stabilisation, to the transcription of various pro-angiogenic
factors, and thus to tumor hyper-vascularisation.³⁰ This hyper-vascularisation of ccRCCs
providing increased tumor oxygenation compared to other cancer types could explain the
differential oxidative stress that we observed. In fact, in VHL-deficient ccRCCs, mitochondrial
respiratory chain activities are decreased depending on HIF stabilization³¹ as a potential
compensatory mechanism to avoid excess ROS production and cancer cell death.³² This makes
cRCC an ideal model to study ROS-mediated cytotoxicity using pulsed-laser irradiation to tip
the balance of ROS homeostasis beyond the tolerance level and induce cancer cell death. When
we analysed each of the five ccRCC xenografts separately, XRCC1 reached the highest levels
of ROS and autophagy markers (Figure S1), suggesting that this model could be particularly
susceptible to ROS-mediated cytotoxicity. We thus chose XRCC1 for further experiments.

To determine the limiting phototoxic intensity for laser irradiation, we irradiated a first
series of XRCC1 xenografts (N = 5) using a femtosecond laser at a mean power of 1000
mW/cm² (234 femtosecond FWHM pulses at 1028 nm wavelength and 60 kHz repetition rate) for 10 minutes. We observed large necrotic areas on the whole irradiated tumor, over 5 mm thick (Figure 3a). We then tested lower laser powers from 400 mW/cm² to 100 mW/cm². Following histological analyses, the mean power of 100 mW/cm² did not induce significant tumor necrosis (Figure 3a and not shown). This was easily monitored by skin temperatures of irradiated areas using a thermal camera (Figure 3b). For all laser powers above 100 mW/cm², the surface temperature excess, ΔT, increased rapidly after 1 to 2 minutes, levelling off between +10°C and +15°C. At a mean power of 100 mW/cm², the temperature increase was at most +5.4°C, corresponding to a skin temperature <42°C, which is acceptable in terms of toxic effect. In the absence of irradiation (control group), there was no skin temperature modification. At a laser mean power of 100 mW/cm², using the same parameters, a repetition rate of 5 kHz did not induce a significantly different effect (data not shown). When we varied the duration of tumor irradiation, up to 30 minutes of continuous irradiation with the same parameters and a mean power of 100 mW/cm², there was no significant difference in skin temperature variation (Figure 3c).

We engineered two different shapes of gold nanoparticles: nanorods and nanoraspberries, excitable at 1028 nm in the second near-infrared window. The gold nanoraspberries have been previously described.³³ Transmission electron microscopy images showed well monodispersed gold nanorods, with a length of 73 ± 9 nm and a thickness of 13 ± 1.5 nm. Their zeta potential was -32.1 ± 0.5 mV. For the gold nanoraspberries, the size and the zeta potential were 25 ± 1.2 nm and -7.3 ± 0.5 mV, respectively (Figure 3d). The two types of nanoparticles were dispersed in aqueous sodium chloride solution with colloidal stability. Both types of nanoparticles absorbed light in the visible-near-infrared window, but over a broad window for the nanoraspberries, while the longitudinal plasmon resonance (λ<sub>LSPR</sub>) of the nanorods in aqueous solution peaked at 1026 nm (Figure 3d), i.e., very close to the laser line
wavelength (1028 nm). We then tested the photothermal performances of the two nanoparticle
types in ccRCC xenografts, using femtosecond pulsed laser and predefined irradiation
parameters, 24h after an intravenous injection of 100 µL, equivalent to 75 µg gold. When the
plateau was reached, we observed that the mean cutaneous temperature increase was +5.2 °C
using gold nanorods while it was only +3.8 °C (*P* < 0.05) using gold nanoraspberries, a value
comparable to that observed in the absence of nanoparticles (Figure 3e); this result is in
accordance with the absorption spectrum of the two nanoparticles (Fig. 3d, right panels), and
with previous data for the nanoraspberries.³³ We therefore retained the gold nanorods (AuNRs)
for further experiments.

The strength of our study was that it optimized the irradiation parameters in order to
decrease toxic effects on normal tissues. We did not induce toxic effect on normal surrounding
skins, even in tumors with high basal levels of oxidative stress, which has good translational
value. The laser power of 100 mW/cm² was the power above which microscopic necrosis was
induced, according to the World Association for photobiomoduLation Therapy WALT
guidelines for laser therapy which recommended 100 mW/cm² for class 3B lasers ³⁴. However,
we observed some biological effects, although limited, in terms of cell death after laser
irradiation, even in the absence of gold nanoparticles and possibly linked to hyperthermia (+3
to 4°C temperature increase) but also to ROS production using laser pulses on a tumor with a
high basal level of oxidative stress. The selection of an appropriate laser system is critical in
the success of ROS-mediated cytotoxicity therapies. We finally retained the following
parameters for further experiments using gold nanoparticles: 234-fs laser pulses at 1028 nm
wavelength and 60 kHz repetition rate, a mean power of 100 mW/cm² (pulse energy 3.85 µJ),
and a 10-mn irradiation duration. We chose a femtosecond pulsed laser instead of a continuous
laser to maximize the generation of ROS, and this is an originality with very little preclinical
data using femtosecond laser pulse irradiation on cancer tissues.⁶, ³⁵ *In vitro*, using a K562
leukaemic cell line, laser pulse irradiation yielded more singlet oxygen generation than the continuous wave mode, with a trend to apoptotic cell death in the pulsed mode.³⁶ These sources emit laser light within extremely short durations, resulting in high peak-power delivery and then multiphoton absorption by the AuNRs, for highly efficient additional ROS production.³⁷ The effects of irradiation on the four complex activities of the mitochondrial chain and ROS production seem closely linked to irradiation parameters, in particular to the laser power and to the wavelength used.³⁸ At 1064 nm, complexes I, III, and IV are activated while complex II is not, possibly leading to higher ROS product generation.

Next, we performed a pharmacokinetic study after a single intravenous injection of AuNRs. Blood, liver, and tumor samples were obtained at different time points after AuNRs administration (Figure 4a). Plasma concentrations peaked at 30 min (140 µg Au/g) with a small decrease over time, and finally, a residual concentration of under 45% on day 14 (Figure 4b). As expected, nanoparticles were detected at high concentrations in the liver as early as day 1, but they slowly decreased over time to 68 µg Au/g at day 7. In contrast, in tumors, gold concentrations were much lower, with a maximum concentration of 10 µg Au/g at day 1, twice as low as early as day 2 (Figure 4b). On the basis of these results, we chose to irradiate the tumors on day 1 after intravenous injection of 75 µg of AuNRs, and to euthanatize the animals on day 3 (Figure 5a). Unexpectedly, when assessing the different ROS products in the irradiated tumors, 4-HNE, nitrotyrosine and 8-oxo-dG showed a significant decrease in mice injected with AuNRs as compared to not only untreated mice, but also mice irradiated without AuNRs injection (*P* < 0.05) (Figure 5b). This was associated with a significant increase in the necrotic area in irradiated tumors of mice injected with AuNRs compared to non-injected mice (60% vs. 42%, *P* < 0.05) and untreated mice (Figure 5c). We then investigated a possible link between this decrease in ROS products and the activation of different signaling pathways. Combination of both irradiation and AuNRs injection induced a significant increase in mRNA expression of
LC3, BECN1 and ATG5, and in protein levels of LC3 (Figure 5d), suggesting activation of autophagy. Although we showed that treatment with irradiation and AuNRs induced a significant decrease in mRNA expression of anti-apoptotic BCL2, and a significant increase in pro-apoptotic markers BID and BAX (P < 0.05), we did not detect any cleavage of the pro-CASPASE-3 protein, demonstrating that apoptosis was not activated in treated mice (Figure 5e). Following irradiation after the injection of AuNRs, an increase in Fe²⁺ accumulation was observed, although it did not reach statistical significance (P = 0.1). In contrast, decreased GPX4 mRNA expression in irradiated tumors was significant (Figure 5f). These results suggested that ferroptosis could be achieved in tumors following treatment. Thus, in our study, the surprising decrease in ROS markers observed after the laser irradiation of mice injected with AuNRs could be explained by the significant increase in cancer cell death and activation of autophagy. The analysis of tumors at much earlier times after irradiation could undoubtedly highlight an early ROS burst before cancer cell death. The induction of most cell death pathways such as apoptosis, autophagy, necroptosis, and ferroptosis in response to the ROS-mediated therapy could have led to the clearance of cancer cells with the highest ROS levels. This is coherent with a reactivation of the mitochondrial respiratory chain in VHL-deficient cancer cells and with a higher sensitivity of ROS mediated cell death of VHL-deficient cells than in VHL-wild type cells.³²,³¹

Interestingly, after irradiation, there was no cell death in the normal skin surrounding the tumor, even in mice injected with AuNRs (Figure 5c). In contrast, in the tumor itself, irradiation alone without nanoparticles at the chosen parameters (mean power of 100 mW/cm² for 10-min), induced some of the biological effects observed with nanoparticles (mainly necrosis, see Figure 5c) but with a gradual intensity between untreated tumors vs. tumors irradiated without AuNRs vs. tumors irradiated after injection of AuNRs. Overall, these results provided evidence of the activation of autophagy and potentially ferroptosis cell-death pathways after irradiation of gold
nanoparticles with femtosecond laser pulses, associated with the disappearance of cancer cells
with high basal levels of ROS.

In conclusion, using femtosecond laser pulse irradiation of tailored gold nanoparticles,
we successfully induced cell death in RCCs with high basal ROS levels. In order to show that
this result is not limited to the RCC tumor type, other tumor types with high basal ROS levels
such as TNBCs should also be tested. One limitation in our study could be the limited anti-
tumor effect observed after a single irradiation. This could be linked to the low gold
concentrations in the tumor after intravenous administration, which could be increased by
optimizing the nanoparticles for active targeting. We have previously reported that a proper
functionalization of gold nanoparticles can significantly increase the intra-tumoral gold
concentration and the anti-tumor effect of the irradiation.⁶ An alternative would be direct intra-
tumoral injection of nanoparticles, highly feasible for deep metastases with current
interventional radiology techniques. Using laser treatments for metastases would replicate the
operational procedure of biopsy by injecting the nanoparticles, inserting the laser fiber, and
applying exposure shortly after injection. As it is already used for focal ablation devices
(cryotherapy, radiofrequency), a temperature control detector at the end of the laser could be
used for temperature monitoring. In addition, gold nanoparticle injections and irradiation could
be repeated over time. We have previously demonstrated the feasibility of weekly tumor
irradiation for four weeks using a femtosecond pulsed laser, without toxicity on surrounding
normal tissues and significant antitumor effect.⁶ Since most cell death pathways are activated
in live cells after a single irradiation, one option would be to combine laser therapy with
systemic treatment such as the use of anti-angiogenic tyrosine kinase inhibitors or
immunotherapies for the treatment of renal cancers. Nevertheless, our study sheds light on the
intricate relationship between ROS and cancer biology. The elevated ROS levels observed in
some tumors highlight their potential as a therapeutic target for ROS-mediated cytotoxicity therapies.

## ASSOCIATED CONTENT

### Supporting information

Material and Methods including human cancer xenograft models, assessment of ROS products and cell-death pathways in tumor xenografts (western blot; immunostainings; RNA extraction and RT-qPCR; lipid peroxidation assay; protein oxidation assay; oxidative DNA damage assay; iron accumulation assay), gold nanoparticles synthesis, laser irradiation and photothermal therapy of RCC xenografts, inductively coupled plasma-mass spectrometry (ICP-MS) and statistical analysis; ethics approval and informed consent; and characterization of several patient clear-cell renal cell carcinomas-derived xenografts in terms of basal oxidative stress and cell death pathway activation (Figure S1).

## AUTHOR INFORMATION

### Corresponding Author

**Guilhem Bousquet**<sup>†</sup> - Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France; AP-HP Hôpital Avicenne, Service d'oncologie médicale, F-93100, Bobigny, France; orcid.org/0000-0001-5594-6694;
Email: [guilhem.bousquet@aphp.fr](mailto:guilhem.bousquet@aphp.fr)

### Authors

**Léa Tourneur**<sup>††</sup> - Université Paris Cité, F-75006, Paris, France; orcid.org/0000-0001-6636-1982; Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France.
**Syrine Gueffrache** - Université Paris-Saclay, CNRS, ENS Paris-Saclay, CentraleSupélec, LuMIn, Gifs/Yvette, France

**Khanh-van Do** - Université Paris-Saclay, CNRS, ENS Paris-Saclay, CentraleSupélec, LuMIn, Gifs/Yvette, France

**Justine Paris** - Université Paris Cité, F-75006, Paris, France; orcid.org/0000-0002-6260-2854

**Yoann Lalatonne** - LVTS, INSERM, U1148, F-93000, Bobigny, F-93000, France; AP-HP Hôpital Avicenne, Service de médecine nucléaire, F-93100, Bobigny, France; Université Sorbonne Paris Nord, F-93430, Villetaneuse, France; orcid.org/0000-0003-2235-6561

**Erwann Guénin** - Sorbonne Université, Université de Technologie de Compiègne, Integrated Transformations of Renewable Matter Laboratory, (EA TIMR 4297 UTC-ESCOM), F-60200, Compiègne, France; orcid.org/0000-0002-7125-9074

**Diaddin Hamdan** - Hôpital la Porte Verte, F-78000 Versailles, France; orcid.org/0000-0002-2212-0287; Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France

**Charlotte Comte** - Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France

**Alexandra Marsocci** - Université Sorbonne Paris Nord, SynKomic research unit, F-93000, Bobigny, France

**Benoît Caron** - ALIPP6, Institut des Sciences de la Terre de Paris UMR 7193, CNRS, Sorbonne Université, F-75005 Paris, France; orcid.org/0000-0001-7051-4339

**Emmanuel Van Glabeke** - Fédération d'Urologie de Seine-Saint-Denis, F-93100, Montreuil, F-93100, France; orcid.org/0009-0009-4607-9854
**Sylvie Marguet** - Université Paris-Saclay, CEA, CNRS, NIMBE, 91191, Gif-sur-Yvette, France; orcid.org/0000-0002-8670-1320

**Christophe Leboeuf** - Université Paris Cité, F-75006, Paris, France; orcid.org/0000-0002-6537-0550

**Bruno Palpant**† - Université Paris-Saclay, CNRS, ENS Paris-Saclay, CentraleSupélec, LuMIn, Gifs/Yvette, France; orcid.org/0000-0003-1376-2533

† and ††These authors contributed equally.

**Author Contributions**

Conceptualization: GB, CL, BP; data analysis: LT, JP, SG, KVD, BC, CC, AM, CL, YL, SM; funding acquisition: BP, GB, SM; investigation: LT, SG, KVD, YL, AM, CL, BC, CC, SM; methodology and statistical analyses: CL, GB, BP, DH; project administration: GB, BP; resources: SM, EVG, YL, BP, EG; supervision: GB, BP, CL; validation of data analyzed: GB, BP, CL, DH, LT; writing – original draft: LT, CL, GB. All authors have given approval to the final version of the manuscript.

**Funding Sources**

This work was supported by the Plan Cancer HEPPROS, Inserm, University Sorbonne Paris Nord, "Plan Cancer" managed by the French ITMO Cancer (no 17CP077-00, project HEPPROS), and by the Public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX-0035, Labex NanoSaclay).

**Notes**

The authors declare no competing financial interest.

**ACKNOWLEDGEMENTS**
We would like to thank Angela Swaine and Sarah Leyshon for the revision of the English language.

The TOC Graphic was created with BioRender.com

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## Figure Legends

**Figure 1.** Diagram of the global effect of oxidative stress in a tumor, (a) with the concept of ROS-mediated cytotoxicity. (b) Cell death signaling pathways (apoptosis, ferroptosis, autophagy, necroptosis) and ROS interaction.

**Figure 2.** Characterization of several patient-derived xenograft models in terms of basal oxidative stress and cell death pathway activation. (a) Clear-cell renal cell carcinomas (ccRCC) and triple negative breast cancers (TNBC) show higher basal levels of ROS products (4-hydroxynonenal (4-HNE), 3-nitrotyrosine and 8-oxo-2'-deoxyguanosine (8-oxo-dG)) than melanomas (Mel), on immunohistochemistry (IHC; only ccRCC is shown), enzyme linked immunoabsorbent assay (ELISA) and western blot quantifications (histograms represent mean 4-HNE/GAPDH ratios ± sem of bands detected in the western blot). (b) Necrotic areas are significantly larger from ccRCCs to melanomas. Necrotic areas observed in hematoxylin-eosin sections (upper panel) are mapped in yellow (lower panel) (X12.5 magnification). Histograms represent the mean percentage for the total of tumor analyzed for each tumor type (N = 20 for ccRCCs, N = 16 for TNBCs, N = 16 for melanomas). (c) Preferential activation of autophagy-related and ferroptosis cell-death pathways in ccRCCs compared to other cancer types. *LC3*, *Beclin1* (*BECN1*) and *ATG5* (autophagy-related gene 5) mRNA were measured using quantitative PCR. LC3A/B proteins were detected by western blot analysis (histograms represent mean LC3A/B/GAPDH ratios ± sem of 14 ccRCCs, 6 TNBC and 3 melanoma). (d) *BCL2*, *BAX* and *BID* mRNA were measured using quantitative PCR. Pro-form and cleaved form of CASPASE-3 protein were detected by western blot analysis. A375 human melanoma cell line treated with doxorubicin was used as positive control (Ctrl) for apoptosis induction. (e) Ferrous iron was measured using ELISA. *GPX4* (glutathione peroxidase 4) mRNA was measured using quantitative PCR. \*P < 0.05; \*\*P < 0.01; ns: not significant.
**Figure 3.** Optimization of irradiation parameters to limit toxic effects on normal tissue. (a) Using 234-femtosecond laser pulses at 1028 nm wavelength and a repetition rate of 60 kHz, and a 10-mn irradiation duration, the variation of the mean power from 1 W/cm² to 100 mW/cm² (pulse energy 3.85 µJ) shows very little necrotic effect at the lowest power. Necrotic areas observed in hematoxylin-eosin sections (left panel) are mapped in yellow (right panel). (b) Monitoring of skin temperature at the site of irradiation is an accurate marker predicting histological toxicity when ΔT is over 6 °C. Dotted line represents the tissue damage temperature cutoff. (c) No temperature increase with duration of time irradiation at a mean power of 100 mW/cm² (234 femtosecond FWHM pulses at 1028 nm wavelength and 60 kHz repetition rate) is observed. (d) Transmission electron microscopy analysis of two different shapes of gold nanoparticles, nanorods and nanoraspberries. Absorption spectrum show that both types of nanoparticles absorb in the visible-near-infrared window, but over a broad window for the nanoraspberries, while the nanorod plasmon resonance (λ<sub>LSPR</sub>) in aqueous solution peaks at 1026 nm. (e) The photothermal performances are better for the nanorods than for the nanoraspberries in ccRCC xenografts irradiated at a mean power of 100 mW/cm² (234 femtosecond FWHM pulses at 1028 nm wavelength and 60 kHz repetition rate) during 10 minutes.

**Figure 4.** Pharmacokinetic study after a single intravenous injection of 75 µg of AuNRs in ccRCC xenografts. (a) Diagrammatic representation of blood and tissue sampling. A single intravenous injection of 75 µg of AuNRs diluted in 100 µL of PBS was performed in ccRCC-xenografted mice. Blood, liver, and tumor samples were obtained at different time points after AuNRs administration, as indicated. Histograms represent mean ± sem of 5 mice at each time point. (b) Gold concentrations over time in blood, liver and tumors.
**Figure 5.** Pulsed-laser irradiation of gold nanorods activates various cell death pathways in ccRCCs with high basal oxidative stress. (a) Diagram of treatment of ccRCC xenografts using pulsed-laser irradiation after intravenous injection of AuNRs. (b) ROS products significantly decrease in irradiated tumors 48h after intravenous injection of AuNRs. ROS products (4-hydroxynonenal (4-HNE), 3-nitrotyrosine and 8-oxo-2'-deoxyguanosine (8-oxo-dG)) were measured using enzyme linked immunoabsorbent assay (ELISA, N = 5 mice for each treatment modality). 4-HNE protein was detected by western blot quantifications (histograms represent mean 4-HNE/GAPDH ratios ± sem of bands detected in the western blot). (c) ROS products decrease is associated with a significant increase in the percentage of necrosis observed in hematoxylin-eosin sections (X12.5 magnification). Histograms represent the mean surface of necrotic areas for each treatment modality (N = 5 mice for each arm). (d) ROS products decrease is correlated with an activation of autophagy cell-death pathway. *LC3*, *Beclin1* (*BECN1*) and *ATG5* (autophagy-related gene 5) mRNA were measured using quantitative PCR. LC3A/B proteins were detected by western blot analysis (histograms represent mean LC3A/B/GAPDH ratios ± sem of bands detected in western blot). (e) Treatments did not induce apoptosis of ccRCC xenografts. *BCL2*, *BAX* and *BID* mRNA were measured using quantitative PCR. Pro-form and cleaved form of CASPASE-3 protein were detected by western blot analysis (histograms represent mean pro-CASPASE-3 ± sem of bands detected in western blot). (f) Potential ferroptosis induction by treatments. Ferrous iron was measured using ELISA. *GPX4* (glutathione peroxidase 4) mRNA was measured using quantitative PCR. NT: not treated; irradiated without nanoparticles; *irrad + Np*: irradiated with nanoparticles; *Np*: nanoparticles.

\*P < 0.05; ns: not significant.
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