# Full Disappearance of PC3-Luc Prostate Tumors Mediated by Hyperthermia Under Low Intensity Ultrasound Application in the Presence of Magnetosomes

Cynthia El Hedjaj, Eric Barret, Imène Chebbi, Raphaël Le Fèvre, Caroline Maake, Franco Guscetti, François Guyot, Jean-francois Aubry, Olivier Seksek, Edouard Alphandéry

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Cynthia El Hedjaj, Eric Barret, Imène Chebbi, Raphaël Le Fèvre, Caroline Maake, et al. Full Disappearance of PC3-Luc Prostate Tumors Mediated by Hyperthermia Under Low Intensity Ultrasound Application in the Presence of Magnetosomes. Advanced Therapeutics, 2025, 8 (2), pp.2400281. 10.1002/adtp.202400281. hal-04917327

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Submitted on 18 Nov 2025

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# Full Disappearance of PC3-Luc Prostate Tumors Mediated by Hyperthermia Under Low Intensity Ultrasound Application in the Presence of Magnetosomes

Cynthia El Hedjaj, Eric Barret, Imène Chebbi, Raphaël Le Fèvre, Caroline Maake, Franco Guscetti, François Guyot, Jean-François Aubry, Olivier Seksek, and Edouard Alphandéry*

Iron oxide nanoparticles have been proposed for magnetic hyperthermia treatment of tumors. However, efficacy depends on the injection of large amounts of such nanoparticles and the equipment is costly. Here, a new thermal cancer treatment is described, in which a tumor containing a low concentration of nonpyrogenic pure iron oxide nanominerals coated with carboxy-methyl-dextran (M-CMD), corresponding to modified magnetosomes, are exposed to ultrasound. Heating PC3 prostate carcinoma cells between 43 and 46°C using ultrasound in the presence of M-CMD resulted in significant necrotic cell death. Furthermore, deposition of M-CMD containing 3 µg of iron per mm³ of tumor in subcutaneous xenografts of PC3-Luc tumors of 150 mm³ followed by 6 to 10 sessions of ultrasound application (1 W cm⁻², 1 MHz) of 10 min each led to a tumor temperature of 43–46°C per session and to total tumor disappearance without regrowth over 6 months following treatment start. Sequential histological analyses of the tumor tissues revealed partial tumor occupation by M-CMD and an increase in cell death over time. Neither lesions, nor magnetosome accumulation were found in microscopic sections of various internal organs collected from treated mice euthanized 6 months after the beginning of the treatment, indicating that M-CMD may not lead to long-term side effects.

## 1. Introduction

Prostate cancer (PCa) is one of the most frequently diagnosed and deadly cancers in men.[1,2] If organ-confined, it is either monitored through active surveillance[3] or treated by surgery[4] or radiotherapy, i.e., external beam radiotherapy (EBR),[5] or brachytherapy with radioactive Iodine 125 seeds implanted into the prostate.[6] Further therapies relying on the focal treatment of PCa have also recently been developed, consisting for example of cryo-therapy,[7-10] photodynamic therapy (PDT),[11-13] thermal-ablation (High Intensity Focused Ultrasound, HIFU),[14-16] Au nanoparticles (NP) heated under laser illumination (Photothermal Therapy, PTT),[17,18] or iron oxide NP exposed to an alternating magnetic field,[19] Although effective, these treatments, particularly radical ones such as prostatectomy[20] or radiotherapy,[21] have significant genito-urinary and gastro-intestinal side effects.

C. El Hedjaj, I. Chebbi, R. Le Fèvre, E. Alphandéry
AlphaOnco (previously Nanobacterie) SAS
36 boulevard Flandrin, Paris 75116, France
E-mail: edouardalphandery@hotmail.com

C. El Hedjaj, O. Seksek
Université Paris-Saclay
CNRS/IN2P3
Université de Paris
IJCLab, Pôle Santé, Orsay 91405, France

E. Barret
Department of Urology
Institut Mutualiste Montsouris
Paris 75014, France

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adtp.202400281

© 2025 The Author(s). Advanced Therapeutics published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/adtp.202400281

C. Maake, E. Alphandéry
Institute of Anatomy
University of Zurich
Winterthurerstrasse 190, Zurich CH-8057, Switzerland

F. Guscetti
Institute of Veterinary Pathology
Vetsuisse Faculty
University of Zurich
Winterthurerstrasse. 268, Zurich CH-8057, Switzerland

F. Guyot, E. Alphandéry
Institut de minéralogie de physique des matériaux et de cosmochimie
Sorbonne Université UMR 7590 CNRS
Université Pierre et Marie Curie
Muséum National d'Histoire Naturelle
4 Place Jussieu, Paris 75005, France

J.-F. Aubry
Institute Physics for Medicine
Inserm U1273
CNRS UMR 8063
ESPCI ParisTech
PSL University
Paris, France
In addition, they have to be carried out in hospital with, in most cases, the need for treatment in an operation theatre. In case of ablative therapy, a careful patient selection is mandatory, as the tumor's location is an important prognostic factor due to the risk of side effects to nearby sensitive structures, such as the urinary sphincter or rectal wall. Access of such treatment is thus rendered difficult for a worldwide large portion of PCa patients, especially those living in poor countries with limited hospital infrastructures or lacking social security or health insurance. To extend treatments to a larger number of PCa patients and improve their benefit/risk ratio, i.e. to achieve full tumor control and reduce the risk of side effects, we describe here a new treatment modality that relies on magnetosomes excited by ultrasound, where ultrasounds generate macroscopic heat and magnetosomes act as local enhancers of the ultrasound excitation.

Magnetosomes are natural iron oxide NP, produced by magnetotactic bacteria (MTB) using nontoxic chemicals. They display a large size leading to ferrimagnetic properties, good crystallinity, well-faceted cuboctahedral shapes, a chain arrangement preventing their aggregation, and a longer tumor residence time than their chemical iron oxide counterparts.[22] Magnetosomes are composed of a chemically stabilized mineral core, from which immunogenic organic material originating from the MTB had been removed. In the present study, nonpyrogenic magnetosome minerals were coated with carboxy-methyl-dextran, and are designated as M-CMD. Furthermore, we opted for an unfocused low-intensity ultrasound (LIU) protocol of 0.2–1 W cm⁻², which is of lower intensities than HIFU (100–10 000 W cm⁻²),[23] and thus associated with lower temperatures. The aim was to prevent potential side effects of thermal-ablation, like coagulative necrosis,[24,25] known from HIFU,[26,27] or magnetic hyperthermia treatment.[28] In addition, LIU could also be produced with simpler and cheaper ultrasound devices than those used in HIFU and achieve a larger focal heating region, i.e. typically ≈1–5 cm³ for LIU compared with 0.5–1 mm³ for HIFU,[29] enabling the coverage of the whole tumor with one application for LIU while several heating spots are often necessary to complete a similar effect with HIFU.[24,25,29,30]

To investigate the anticancer potential of our LIU/M-CMD combined treatment, we here analyze its effects both in vitro on PC3 cells and in vivo on mice bearing subcutaneous PC3-Luc prostate adenocarcinoma xenografts. While nonluminescent PC3 cells were used for in vitro studies to avoid interferences with the fluorophorescence of the cell death assays, their luminous counterparts with similar properties were employed for in vivo studies to detect non palpable tumors. For in vivo studies, mice received at the center of their tumor a dose of 3 µg of M-CMD per mm³ of tumor. Such a dose is ≈8 times lower than the magnetosome dose previously used to treat glioblastoma multiforme (GBM) by magnetic hyperthermia in mice.[22] The ultrasound intensity and associated tumor temperature that yield optimal antitumor efficacy were determined. Histological analyses of tumor tissue after the different ultrasound sessions, and liver, spleen, lung, brain and kidney tissues collected 6 months after the first treatment, were performed to monitor the distribution of M-CMD and type and distribution of lesions in the tumors.

# 2. Results and Discussion

## 2.1. M-CMD Synthesis and Characterization

During the pregrowth step, MTB were cultivated in the absence of iron, leading to the amplification of bacteria lacking magnetosomes (Figure 1a). In the growth-step, MTB further divided in a growth medium fed with an iron-rich fed-batch medium, and produced intracellular magnetosomes (Figure 1b). Both stages presented a spirillum-like morphology and length of 3.75 ± 1.5 µm, supporting the idea that bacterial integrity is preserved during the two steps of bacterial growth. Then, magnetosomes were extracted from MTB by alkaline lysis and isolated from bacterial debris with a magnet, leading to magnetosomes arranged in chains (M-Ch) (Figure 1c). Subsequently, M-Ch were purified through heating in an oven to remove most of the remaining organic material by combustion, yielding pure inorganic uncoated magnetosome minerals (M), assembled as individual aggregated magnetosomes (Figure 1d). To be stabilized, magnetosome minerals were coated with CMD to yield M-CMD, which appear in the form of reconstructed well-dispersed magnetosome chains as can be seen in Figures 1e,f. Further analysis of M-CMD TEM images demonstrated that dried M-CMD were mono-dispersed with average core sizes of 36.4 ± 8.1 nm and coating thicknesses of 2.5 ± 0.5 nm (Figure 2a), and were homogeneously distributed without aggregation (Figures 1e,f). CMD-coating clearly appeared as a light gray 2–3 nm thick layer surrounding the magnetosome mineral core, which appeared with a dark gray cuboctahedric shape in the TEM images of Figure 1e, and is not present for uncoated magnetosomes (Figure 1d). CHNS Elemental measurements carried out on magnetosomes enable a follow-up of the magnetosome organic composition at the different steps of magnetosome fabrication (Figure 2b). After their extraction from MTB, M-Ch display relatively large percentages of carbon (25%) and nitrogen (2.5%), which decrease to 0.1% carbon and 0.02% nitrogen in noncoated magnetosomes (M) following heat treatment. Then the percentage of carbon increases again, up to 2.5% in M-CMD due to CMD organic coating being added to the magnetosome mineral core. Additional results support the idea that M-CMD in suspension are injectable. Firstly, in water suspension, M-CMD maintain a stable absorbance at 480 nm during 7'000 seconds in contrast to uncoated magnetosome minerals (M) that rapidly yield vanishing absorbance due to sedimentation (Figure 2c), indicating that M-CMD are stable during a period of time that is sufficiently long to enable their injection as a homogenous suspension. Secondly, M-CMD display a sufficiently low endotoxin concentration of 90 EU mL⁻¹ mg⁻¹ (Figure 2d) to be considered as nonpyrogenic. While endotoxins are present in a relatively large quantity in CM suspensions at 6'000 EU mL⁻¹ mg⁻¹ (CM), originating from MTB external membrane, they reach a very low concentration of 80 EU mL⁻¹ mg⁻¹ in uncoated magnetosomes (M) due to the heat treatment. They are maintained at such a low level in M-CMD by having M coated with CMD under sterile conditions. Thirdly, the zeta potential of M-CMD (Figure 2e) is negative over a large range of pH values, i.e. pH > 3.2, which could stabilize M-CMD in suspension due to electrostatic repulsions.[31] Fourthly, ICP-MS measurements (Figure 2f) carried out on dissolved
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Transmission electron microscopy images of magnetotactic bacteria and magnetosomes obtained at the various steps of the magnetosome fabrication process, i.e of magnetotactic bacteria collected at the end of the pregrowth step with iron-free growth medium, a), of magnetotactic bacteria harvested after the growth step in the presence of iron, b), of chains of magnetosomes (M-Ch) extracted from magnetotactic bacteria, c), of purified magnetosome minerals (M) from which most bacterial organic material has been removed, d), of magnetosome minerals coated with carboxy-methyl-dextran (M-CMD) that are organized in chains with a magnification showing a typical chain of M-CMD, e), and with a lower magnification displaying an assembly of several chains of M-CMD with a homogenous distribution, f).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Histogram showing the size distribution of M-CMD deduced from TEM measurements carried out on 300 magnetosomes, a), CHNS measurements showing the evolution of the percentage in mass in carbon and nitrogen present in the various types of magnetosomes obtained at the different steps of the magnetosome production, i.e. chains of magnetosomes (M-Ch), magnetosome minerals (M), and magnetosome minerals coated with carboxy-methyl-dextran (M-CMD), b), variation of the absorption of a suspension of M-CMD and M homogenized by shaking in pyrogen-free water, measured at 480 nm, as a function of time, normalized by the initial value of the absorption, representing the percentage of stability of these suspensions, c), endotoxin concentrations, measured by the LAL assay in M-Ch, M, and M-CMD, d), Zeta potential of suspensions containing M and M-CMD measured as a function of pH, e), percentage of iron vs. other metals in M-CMD, as measured by ICP-MS, f).</figcaption></figure>

M-CMD indicate that M-CMD are composed of a large percentage of iron of 96%, as estimated through the formula $%Fe = m_{Fe} \% \sum_{i=1}^{n} m_{mi}$, where % Fe is the percentage in mass of iron in M-CMD, $m_{Fe}$ is the quantity of iron in M-CMD, and $\sum_{i=1}^{n} m_{mi}$ is the sum of the masses of all metals present in M-CMD. The remaining metal in the magnetosome composition consists of

2.8% of magnesium, 0.8% of silica, 0.1% of aluminum, 0.1% of chrome and 0.2% of the other elements (Pb, Ti, Mn, Zn, Ni, Ge, Sr, Zr, Li, Ba, Mo and Cu). Taken altogether, these results indicate that M-CMD are stable in suspension, nonpyrogenic, pure in iron, and therefore display favorable properties for their injection.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Temperature course during ultrasound exposure of PC3 cells incubated with cell medium only, a), or with 500 µg mL⁻¹ in iron of M-CMD, (b), for 3 hours, and exposed to ultrasounds with an intensity of either 1 W cm⁻² at a frequency of 1 MHz for 10 and 20 min. Viability of PC3 cells 24 hours after treatment as above in dependence of the duration of ultrasound exposure c). Propidium iodide/Annexin-V flow cytometric analysis of PC3 cells by being incubated (or not) with 500 µg mL⁻¹ of M-CMD (cyan color), or by being exposed to LIU of 1 MHz and 1 W cm⁻² in the absence of M-CMD (green color) or in the presence of 500 µg mL⁻¹ of M-CMMD (red color). The number of PC3 cells counted by flow cytometry as a function of the fluorescence signal (ECD canal), measuring propidium iodide fluorescence intensity d). All data are reported as mean ± SD, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 (ANOVA, see Section 1.3.7).</figcaption></figure>

## 2.2. In Vitro Heating and Antitumor Efficacy of M-CMD in PC3 Prostate Cancer Cells Exposed to Low Intensity Ultrasound

In vitro studies were carried out to examine whether LIU/M-CMD treatment leads to tumor cell death, and to preliminarily determine its type. For that, PC3 cells were incubated (or not) with 500 µg mL⁻¹ of M-CMD during 24 hours and exposed to LIU of 1 W cm⁻² and 1 MHz for 10 or 20 minutes. Heating curves derived from these experiments are presented in **Figures 3a,b**. They are relatively similar in the presence and in absence of M-CMD, and are characterized by an initial phase of temperature increase from 25°C to 45–46°C (which represents the whole treatment for 10 minutes of LIU application) followed by a second period of 625 seconds during which the temperature is stabilized (for 20 minutes of LIU application). This translates into a statistically significant increase in CEM 43 from 8 minutes for 10 minutes of LIU application to 81 minutes for 20 minutes of LIU application (Figure S2, Supporting Information), suggesting a biologically relevant difference in the thermal dose. This further resulted in an increase of tumor cell death from 0% without M-CMD to 30% with M-CMD (Figure 3c).

Since hyperthermia-induced cancer cell death in the presence of nanoparticles is often correlated with nanoparticle cellular internalization,[32] we have studied if this applies for M-CMD as well. For that, the quantity of iron originating from magnetosomes was estimated with ICP-MS in treated cells. While the amount of internalized M-CMD was considerable before LIU treatment (26 ± 3.5 pg cell⁻¹) it did not increase following LIU application at 1 W cm⁻² remaining at a similar value of 27.4 ± 4.1 and 29.9 ± 5.9 pg cell⁻¹, respectively (Table S2, Supporting Information). Furthermore, since such quantity represents a rather small percentage of the volume of a tumor cell occupied by magnetosomes, i.e., ≈2.10⁻⁶%.[33,34] Intracellular space may remain available for more magnetosomes to be internalized in it. However, heat produced by LIU might fragilize tumor cells, hence underscoring the efficacy of M-CMD internalization.

Furthermore, to examine the type of cellular death induced by the treatment, cells treated as in Section 1.3.2 were stained with propidium iodide (PI) and Annexin V-FITC. The intensity of fluorescence and the number of PC3 cells stained (or not) was determined by flow cytometry (Section 1.3.4). For all treatment conditions, 100% of PC3 cells were not labeled in the FITC channel
(data not shown), indicating that the LIU/M-CMD treatment does not trigger tumor cell death via apoptosis. By contrast, Figure 3d shows that the PI signal, which is indicative of necrosis, is observed for cells treated by the LIU/M-CMD treatment (28.2% of cells are necrotic), while it is absent for nonheated cells (PC3 cells alone or PC3 cells with M-CMD) and for those heated by LIU without M-CMD (0.58% of cells are necrotic). Taken altogether, these results indicate that the LIU/M-CMD treatment results at least partly in a necrotic tumor cell death, and that heating cells by applying LIU in the absence of M-CMD is insufficient to yield such a cell death mechanism.

## 2.3. Antitumor Efficacy in Mice Bearing PC3-Luc Xenografts treated by Intra-Tumor Administration of M-CMD Followed by Several Ultrasound Sessions

In nanomedicine, the main method of hyperthermia treatment on tumors is to vectorize the NPs or inject them directly into the tumors and then apply an excitation source e.g. laser for Au NP.[35] or alternating magnetic field for iron oxide NP.[36] In those cases, the produced heat is highly dependent on the NP dose, i.e. the magnitude of produced heat increases with increasing amount of NP exposed to the source of excitation.[37] Such behavior can be problematic when the NP dose in the tumor varies over time, e.g., when NP leave the tumor. Here, we chose an ultrasound-based heating method, yielding a sufficiently large heated volume (≈1–5 cm³) to cover the whole tumor instead of the sole NP region reached with the more focused heating methods mentioned above. However, the area exposed to ultrasound remained below the surface of the transducer of diameter 3.6 cm used for LIU treatment, avoiding systemic heating of the whole mouse, as was evidenced by the absence of rectal temperature increase as measured during the various sessions of ultrasound application (Figures 4a,c,e,g and Figures S3, S4, and S5, Supporting Information). Furthermore, our ultrasound-based treatment yielded heating properties, in particular saturating temperatures, that were mainly dependent on the intensity of the applied ultrasound instead of on NP presence in the tumor (Figures 4a,c,e,g and Figures S3, S4, and S5, Supporting Information). We demonstrate that the tumor temperature can be adjusted by tuning the ultrasound intensity, while the presence of M-CMD in the tumor results in an enhanced antitumor efficacy above a certain tumor temperature threshold, which corresponds to the temperature achieved during moderate hyperthermia.

We start by analyzing the groups receiving intratumorally at D0 either glucose only (group I) or 3 µg mm⁻³ of M-CMD (group III) without ultrasound application. In these cases, the tumor and whole mice followed the same behavior of slight temperature decrease of ≈4°C during the anesthesia performed during glucose and M-CMD injection (Figure 4a and Figure S6a, Supporting Information). For both groups, the tumors displayed a similar exponential increase of tumor growth from 100 mm³ at D0 to 1100 mm³ at D12-D17 (Figure 4b and Figure S6b, Supporting Information) followed by euthanasia (endpoint) of these mice over short times between D8 and D57 (Figure 4b and Figure S6b, Supporting Information), an outcome attributed to the aggressiveness of PC3-Luc prostate tumors and to the absence of antitumor efficacy for M-CMD not exposed to ultrasound. Indeed,

the statistical analysis showed the absence of significant differences between these groups (I and III) for the survival rates using the log-rank test (Table S3, Supporting Information) and for the tumor volume using the Mann- and Whitney test (Table S4, Supporting Information).

When ultrasound of low intensity (0.2 W cm⁻²) was applied on tumors in the absence of M-CMD (group VIII) or in presence of M-CMD (group VI), the tumor temperature increased during the first 200–300 seconds from 30–32°C to a stabilizing temperature, Tsat of ≈34–38°C, and then persisted at Tsat during the remaining time of the ultrasound session (Figure 4c and Figure S6c, Supporting Information). Tumors grew exponentially, and mice were euthanized as early as at D17 (Figures 4d and Figure S6d, Supporting Information). Such outcome was similar to that observed in groups I and III as shown in statistical analysis in Tables S3 and S4 (Supporting Information). This reveals an absence of sustained antitumor activity for ultrasound treatment at 0.2 W cm⁻² that can be attributed to a too low heating temperature reached during the ultrasound heating sessions, i.e. Tsat < 38°C (Figure 4c and Figure S6c, Supporting Information).

As the ultrasound intensity was increased to 0.5 W cm⁻², Tsat increased up to 41–44°C (Figure 4e and Figure S6e, Supporting Information), resulting in partial antitumor efficacy for PCa xenografts either injected or not with M-CMD (groups V and VII). This treatment led to tumor growth retardation, i.e. the tumor growth rate was calculated at 29 mm³ per day at 0.5 W cm⁻² (Figure 4f and Figure S6f, Supporting Information) compared to 60 mm³ per day for the groups I and III (Figure 4b and Figure S6b, Supporting Information). In the former groups, however, the antitumor efficacy was not sufficient to prevent tumor growth and the tumor volume increased from 100 mm³ at D0 to 1100 mm³ at D22-D38 (Figure 4f and Figure S6f, Supporting Information). Consequently, mice had to be euthanized after D20 and D30 in the absence and presence of M-CMD, respectively (Figures 4f and Figure S6f, Supporting Information). Indeed, mice of group V (M-CMD + US 0.5 W cm⁻²) had a median survival of 41.5 days, which is longer than in group I (21 days), but this difference was not statistically significant (Tables S3 and S4, Supporting Information). Tumor growth delay could be achieved with other types of nanomedicine, e.g. when superparamagnetic iron oxide nanoparticles introduced into prostate tumors grafted under the skin of rats were exposed to an alternating magnetic field of 100 kHz and 0–18 kHz m⁻¹, but this required a higher heating temperature of more than 54°C.[38]

By exposing PCa xenografts to ultrasound of higher intensity, i.e. 1 W cm⁻², the tumor temperature reached a higher value of Tsat ≈ 43–45°C both in the absence (group II) and presence of M-CMD (group IV), with Tsat being reached after 200 s, i.e. more rapidly than for lower ultrasound intensities (Figure 4g and Figure S6g, Supporting Information). Consequently, a more robust antitumor activity was achieved at 1 W cm⁻² than at 0.2 W cm⁻² and 0.5 W cm⁻². In the absence of M-CMD injection, the antitumor activity however remained partial, resulting in a tumor growth rate of 16 mm³ day⁻¹ (Figure 4h and Figure S6h and Tables S4 and S5, Supporting Information). However, most mice were either euthanized prematurely because the endpoint was reached, or they displayed tumor disappearance followed by tumor regrowth from D30 (Figure 4h, Figure S6h and Tables S6 and Table S3, Supporting Information). A unique mouse was cured
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Intratumoral and rectal temperatures measured after intratumoral glucose or M-CMD administration (mice groups I and III, respectively) without ultrasound application, a), or during the third session of ultrasound application carried out at 0.2 W cm⁻² (mice groups VI and VIII), c), 0.5 W cm⁻² (mice groups V and VII), e), or 1 W cm⁻² (mice groups IV and II), g), in the presence or absence of 3 µg mm⁻³ of intratumoral M-CMD, respectively. Variation of the tumor volumes of mice belonging to the groups as above following all sessions of ultrasound application. Values for groups I and III, b), groups VI and VIII, d), groups V and VII, f), and groups IV and II, h). The mean value of the measurements in all animals and standard deviation are indicated. All data are reported as means ± SD, * *p*-value < 0.05, ** *p*-value < 0.01, *** *p*-value < 0.001 (Mann–Whitney, see Section 1.4.7).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Variation of the tumor volume over the course of the treatment with representative photographic and BLI images of one mice belonging to group IV treated by intratumoral administration of 3 µg mm⁻³ of M-CMD followed by 10 sessions of ultrasound application of intensity 1 W cm⁻² and frequency 1 MHz, a) and of one mice belonging to group II treated by intratumoral glucose administration followed by 10 sessions of ultrasound application of intensity 1 W cm⁻² and frequency 1 MHz, b). Survival curves of mice treated by intratumoral administration of 3 µg mm⁻³ M-CMD without further treatment (group III), or M-CMD followed by ultrasound applications of 1 W cm⁻² (group IV), 0.2 W cm⁻² (group VI), or 0.5 W cm⁻² (group V), c), and of mice given glucose only intratumoral administration (group I), or glucose followed by 10 sessions of ultrasound application of 1 W cm⁻² (group II), 0.2 W cm⁻² (group VIII), 0.5 W cm⁻² (group VII), d). Survival rate was analyzed using Kaplan Meier, \* *p*-value < 0.05, \*\* *p*-value < 0.01, \*\*\* *p*-value < 0.001 (Log rank, see Section 1.4.7).</figcaption></figure>

and survived 225 days following the beginning of treatment, possibly due to a smaller initial tumor volume than in other mice. In contrast, when the application of ultrasound of 1 W cm⁻² was carried out in the presence of M-CMD in the tumor, complete tumor disappearance was obtained without tumor regrowth following 6 to 10 ultrasound sessions (Figure 4h and Figure S6h and Tables S4 and S5, Supporting Information). These good efficacy results are further highlighted in Figures 5a,b, which represent the evolution of the tumor volume and tumor BLI over the course of the treatment for two representative mice treated at 1 W cm⁻² without M-CMD (Figure 5b) or with M-CMD intratumoral injection (Figure 5a). In the absence of M-CMD, the tumor volume decreased from 200 mm³ at D0 to a value close to 0 between D10 and D30, and then increased exponentially after D32 (Figure 5b). The improvement in the antitumor efficacy observed between 0.5 W cm⁻² and 1 W cm⁻² corresponded to an increase in the CEM 43 value averaged over all heating sessions and mice, which was below 10 minutes for 0.5 W cm⁻² and above this value for 1 W cm⁻² (Figure S7, Supporting Information).

Hence, this treatment condition led to transient antitumor activity, as further highlighted by the mouse survival curve of group II where mice started to be euthanized at D35 (Figure 5d). In the presence of M-CMD, mice belonging to group IV display a similar decrease in tumor volume between D0 and D10 as observed in group II. However, tumor regrowth was not observed for group IV and both the tumor volume and tumor BLI remained at values close to 0 between D10 and D120 (Figure 5c). The absence of primary tumor regrowth among mice of group IV further resulted in a survival rate of 100% during 60 days following the beginning of the treatment (Figure 5c, Suppl Tables S3, S6 and S7). Other ultrasound-based treatments of rats or mice bearing sarcoma or breast tumors, administered with sonosensitizers such as SF-1, HMME, or SPION, and exposed to ultrasound, in combination (or not) with the use of PD-L1 blockade checkpoint inhibitor or laser irradiation, led to tumor regression. However, a long-term full disappearance of the primary tumor, as is demonstrated here, was not shown in those experiments.[39,40]
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Microscopical images of tumor tissues (Perls staining) from mice treated by M-CMD intratumoral administration followed by sessions of ultrasound application. Microscopical images of histological slides of tissues located in the region of the treated tumor collected 2 hours after the first session of ultrasound application, a), 1 day after start of treatment, b), 2 days after start of treatment, c), 7 days after start of treatment, d), 21 days after start of treatment, e), or following euthanasia 6 months after the beginning of the treatment, f). Higher magnification of typical sections of the tumor region displaying both absence of neoplastic cells and magnetosomes in (e') and (f').</figcaption></figure>

## 2.4. An Optimal Antitumor Activity Achieved for Moderately Heated Prostate Tumors (at 45°C) infiltrated heterogeneously with Magnetosomes

Heat appears as an important factor for the antitumor effect. Here, it is generated through a transformation of acoustic energy into thermal energy.[33] Indeed, the sole administration of magnetosomes into tumors without application of ultrasound does not induce any damage on the tumor. In addition, to achieve optimal antitumor efficacy, it seems that a temperature threshold must be reached, i.e. around 43–46°C. Indeed, below this temperature, the antitumor effect is weaker.

Furthermore, magnetosomes act as sonosensitizers, i.e. they yield an enhanced antitumor activity under optimized conditions of ultrasound heating. Therefore, magnetosomes are classifiable as a medical device. For mice belonging to group IV having received the combined M-CMD/LIU treatment, a crust (3.6 cm of diameter) initially appeared after the 3rd and the 4th session, and disappeared completely two weeks following the last heating ses-

sion, leaving a scar. After 6 months, neither tumor regrowth nor scar was observed on cured mice backs.

Next, we analyzed whether direct contact between magnetosomes and tumor could induce tumor cell destruction or if the demise of the cells can also take place at some distance from the magnetosomes, as it is the case in radiotherapy through the by-stander effect.[41,42] We have led for treatment group IV a histological study of tumor tissues collected after the various ultrasound sessions S1 (Figure 6a), S2 (Figure 6b), S3 (Figure 6c), S4 (Figure 6d), S10 (Figure 6e), and of the skin tissue located in the region of previous tumor growth harvested 6 months following the beginning of the treatment (Figure 6f). The sections were stained with an iron-revealing Perls staining. As illustrated in Figure 6 and Figure S8 (Supporting Information), a representative section (5 sections were analyzed per block) of low-magnification histological Perls stains showed an inhomogeneous, partly extensively disseminated, partly focal distribution of the magnetosomes up to S4. These observations are in accordance with previous studies with alternating magnetic field
<figure><img src="image_9.png" /><figcaption>**Figure 7.** Microscopical images of tumor tissues (H&E staining) from mice treated by M-CMD intratumoral administration followed by 1 session of ultrasound application at 1 W cm⁻², a), or 4 sessions of ultrasound application, b). (a') and (b') represent larger magnifications of necrotic regions in (a) and (b), respectively.</figcaption></figure>

showing that magnetosomes are very effective to treat heterotopic and orthotopic glioblastoma tumors[22,43] although they are not homogeneously distributed all over the tumor tissue. At the end of the treatment, i.e. at S10 and 6 months after the beginning of the treatment, magnetosomes were no longer observable in the tissues collected from the original tumor location (Figure 6e,f), indicating gradual magnetosome depletion from the tumor during the course of the treatment. At the end of the treatment, a complete absence of magnetosomes was observed in the skin (Figure 6f and Figure S9a, Supporting Information). Under our conditions of application of LIU, it is not obvious that magnetosome cellular internalization is favored at D0, but at later time points magnetosomes mostly appear to be localized intracellularly (except for necrotic areas). Considering the heterogeneous distribution of the magnetosomes in the tumor tissues, it appears that magnetosomes don't necessarily need to be all in direct contact with tumor cells or internalized inside these cells to yield op-

timal antitumor efficacy. This result is of particular interest since it may be challenging to achieve NP internalization in 100% of tumor cells in a patient. The condition highlighted here to reach full tumor disappearance, i.e. heterogenous infiltration by the magnetosomes, seems much easier to achieve clinically.

Histological assessment of the tumor morphology by H&E-stained sections, carried out after 1 (Figure 7a) and 4 heating session(s) (Figure 7b), showed a focally-extensive viability loss of tumor cells, progressing over time and ending in overt extensive necrosis after S4 in animals exposed to 1 W cm⁻² ultrasound (Figure 7b). No obvious qualitative differences were visible between the groups IV and II (injected vs. not injected with M-CMD and exposed to 1 W cm⁻² ultrasound, respectively). Representative histological images of the treatment groups are shown in Figures S10–S16 (Supporting Information). Tumor cell death could be attributed to a combined effect, which is due to heat and the presence of magnetosomes in the tumor. Indeed, while
relatively similar temperatures are reached with and without magnetosomes during each session at 1 W cm⁻² (Figures S3–S5, Supporting Information), antitumor efficacy is much more pronounced in the presence than absence of magnetosomes (Figure 4h). Furthermore, for mice treated in the absence of ultrasound application or for those exposed to ultrasound of intensity lower than 1 W cm⁻², i.e. 0.2 W cm⁻² and 0.5 W cm⁻², tumor cell death was either absent or much less pronounced than at 1 W cm⁻² (Figures S10–S16 and Table S3, Supporting Information), indicating that the application of ultrasound of 1 W cm⁻², which yields a tumor temperature of 43–46°C, is necessary to induce sufficient cellular death and result in full tumor disappearance.

**2.5. Safety of the Treatment**

None of treated mice, with/without magnetosomes injections and with/without ultrasound application (groups I to VIII), showed detrimental clinical signs, including loss of body weight or changes in behavior.

To study the safety of the treatment, two mice belonging to group IV treated under the conditions of optimal treatment efficacy were euthanized 6 months following the beginning of the treatment, and different organs of these mice (liver, brain, kidney, lung, spleen) analyzed histopathologically. As shown in **Figure 8**, the different organs did not present any sign of lesion, indicating that the treatment does not lead to any long-term side effects observable by histology. Furthermore, no accumulations of iron were observed in any of these organs, suggesting that magnetosomes are eliminated following treatment. The skin tissue located at the tumor site, which was harvested 6 months following the start of the treatment, did not display neither tumor cells nor magnetosomes (Figure S9, Supporting Information), indicating their complete removal from the initial tumor site. H&E stained slides of these tissues show reconstitution of the skin despite the development of temporary crusts on the skin during the treatment. Thus, treatment seems to be safe from a long-term perspective at least for subcutaneous types of cancer. Additional toxicity studies (more details in a publication in progress) were carried out to evaluate M-CMD safety profile, indicating first that according to the criteria of ISO 10993–5 M-CMD are not cytotoxic at high concentrations (up to 1 mg mL⁻¹) to L929 cell lines. M-CMD genotoxicity studies were evaluated according to ISO 10993-21, on V79-4 cell lines, revealing that the percentages of micro-nucleated cells after treatment with metabolized M-CMD (with S9 liver extract mix that stimulates cell's metabolism, Merck) or un-metabolized M-CMD are similar to those obtained with untreated cells. These results show an absence of the genotoxicity effect of M-CMD on V79-4 cell lines. Taken altogether, these data are in line with previously published results, which report an absence of observable magnetosome toxicity.[44–47]

**2.6. Advantages and Innovative Aspects of our Treatment**

The advantages of the treatment presented in this article rely on three innovative aspects, which are not found in other focal therapies of prostate tumors such as high intensity focused ultrasound, irreversible electroporation, and cryotherapy.[48]

Firstly, we chose a moderate heating temperature with the following properties: i) it is sufficient to destroy tumor tissue but not too high to avoid damaging surrounding healthy tissue, ii) it enables heating a sufficiently large area to encompass the whole tumor within a single ultrasound application, iii) it allows indirect biological mechanisms to destroy tumor tissue at a certain distance from the magnetosomes, hence making a complete infiltration of the tumor tissue with magnetosomes unnecessary, which is clinically difficult to achieve.

Secondly, heat is essentially produced macroscopically by ultrasounds, making it rather independent of the magnetosome concentration. Thus, lower nanoparticle concentrations than those used in magnetic hyperthermia can be used, i.e. our nanoparticle concentration of 3 µg mm⁻³ is ≈20 lower than that used to treat prostate tumors with chemical iron oxide nanoparticles using magnetic hyperthermia.[45] At the same time, antitumor efficacy of our treatment can be preserved over a long period of typically a few weeks following nanoparticle injection, i.e. even when a small quantity of magnetosomes remains in the tumor.

Thirdly, on the one hand, magnetosomes are biocompatible, which is due to their natural method of fabrication that excludes toxic compounds, to their high level of iron purity, i.e. their composition in heavy metals other than iron is very low, the removal of endotoxins and other organic biological material. On the other hand, they are stable, due to the presence of a synthetic coating of CMD that surrounds their inorganic iron oxide mineral core that prevents their aggregation.

Fourthly, our treatment is relatively straightforward to implement since it does not require the use of the operation theatre or general anesthesia. It could therefore potentially be carried out on an outpatient basis or under the simple consultation of an urologist.

**3. Conclusion**

In conclusion, we have developed a new method of tumor treatment that operates through the injection of magnetosomes in the tumor followed by several 10-minute heating sessions at 43–46°C under ultrasound application. Such a method displays a series of advantages. Firstly, the tumor heating temperature is unaffected by the presence of nanoparticles, i.e. a desired heating temperature can be reached by adjusting the values of the parameters of the applied ultrasound independently of the nanoparticle intratumoral distribution, which can evolve during the course of the treatment. Secondly, the treatment is fully efficient, leading to the complete disappearance of the tumor in all treated mice. Thirdly, the reconstituted magnetosomes, which are used as sonosensitizers to increase the antitumor efficacy triggered by the heat-generating ultrasound, display a series of advantageous properties such as a natural origin and a synthesis without the use of toxic products, a nonpyrogenicity ensured by a double detergent/heat based treatment, a high iron purity, and an arrangement in chains that allows magnetosomes to remain in the tumor for a time period that is sufficiently long for the treatment to reach its full efficacy, as shown in another study when mouse glioblastoma were efficiently treated by exposing magnetosomes to an alternating magnetic field.[22] To pursue the work described in this study, we plan to improve the way in which the tumor is exposed to ultrasound. For that, we might first apply
<figure><img src="image_10.png" /><figcaption>**Figure 8.** H&E-stained histological slides of various organs of a mouse, i.e. liver, brain, kidney, lung, spleen, collected 6 months after a treatment consisting in intratumoral administration of 3 µg mm⁻³ of M-CMD followed by 10 sessions of ultrasound application at 1 W cm⁻² and 1 MHz.</figcaption></figure>
ultrasound sequentially with the help of a Programmable Logic Controller (PLC) to stabilize the tumor temperature at 45°C during treatment. Secondly, we might add a water-cooling system to the transducer or use a specific transducer configuration to prevent overheating the region of contact between the gel and skin and hence to avoid the formation of a crust. The study presented here complements our previous work describing various magnetosome applications in the medical field, including fabrication of very pure magnetosomes with a metallic composition of 99.9% in iron,[41] magnetic hyperthermia treatment of tumors,[42] fluorescence imaging in a biological environment,[49,50] and multiparametric detection in a magnetic sensor.[51]

## 4. Experimental Section

**Synthesis and Characterization of Magnetosome Minerals Coated with Carboxy-Methyl-Dextran (M-CMD):** Growth of MSR-1 Magnetotactic Bacteria – briefly, MSR-1 magnetotactic bacteria (MTB) were purchased from DSMZ (DSM-6361, Braunschweig, Germany). In a preamplification step, we cultured an aliquot of 1 mL containing $10^8$ MTB in a pregrowth medium devoid of iron for 9 days in the presence of $O_2$ up to an optical density measured at 565 nm ($OD_{565}$) of ≈1. The medium contained, in one liter, 2.6 g of lactic acid, 0.4 g of $NH_3$, 0.1 g of $MgSO_4 \cdot 7H_2O$, 0.5 g of $K_2HPO_4$, 0.5 mg of $FeSO_4 \cdot 7H_2O$, 13.2 mg of $CaCl_2$, 0.02 g of Biotin, 0.4 g of calcium pantothenate, 0.002 g of folic acid, 2 g of inositol, 0.4 g of nicotinic acid, 0.2 g of p-aminobenzoic acid, 0.4 g of pyridoxine HCl, 0.2 g of riboflavin and 0.4 g of thiamine HCl (vitamin B1). In a second step, preamplified MTB were further amplified in a fermenter containing the same growth medium as above under fed-batch conditions using an acid solution containing, in one liter, 100 g of lactic acid, 4.8 g of $NH_3$, 2.4 g of $MgSO_4 \cdot 7H_2O$, 6 g of $K_2HPO_4$, 7 mg of $FeSO_4 \cdot 7H_2O$, 180 mg of $CaCl_2$, and the same 9 vitamins as above and an iron source containing 2 g of $FeCl_3 \cdot 6H_2O$, which was inserted in the growth medium to feed the bacteria with iron, promote magnetosome formation while maintaining the pH of the growth medium at 6.9. During this second step, bacterial growth was sustained by bubbling $O_2$ in the growth medium with an air compressor maintaining the $O_2$ concentration in the medium below 0.1% to enable magnetosome synthesis. After 4 days of the second step, MTB reached an $OD_{565}$ ≈ 10 and contained ≈ 25 mg of magnetosomes in iron per liter of the growth medium, as deduced by an iron assay. The growth conditions are described in more detail in Berny et al., 2020.[52]

Preparation of nonpyrogenic uncoated magnetosome minerals – following MTB bacterial growth, the suspensions of MTB were concentrated by factor 5 using a tangential filtration column (PES 0.2 µm, Repligen). Then, bacteria were lysed by mixing with KOH at 80 °C for 1 h and magnetosomes were separated from bacterial debris using a strong Neodymium magnet positioned against the glass vessel filled with the lysed MTB suspension and KOH. The lysed MTB/KOH liquid suspension was then replaced by PBS using a serological pipette. This step was repeated to remove PBS with water. Hence, most organic material originating from the MTB was removed. Magnetosomes thus obtained, were concentrated, lyophilized, and further purified by heating in the presence of $O_2$ in a muffle oven to obtain sterilized magnetosome minerals.

Preparation of magnetosome minerals coated with carboxy-methyl-dextran – suspensions of sterile magnetosome minerals were sonicated for 20 hours at 25 kHz under sterile conditions at an iron concentration of 2 mg mL⁻¹ in the presence of a carboxy-methyl-dextran (CMD) solution at 240 mg mL⁻¹ and pH 4.5. Coated magnetosome minerals (M-CMD) were centrifugated at 3380 × g for 45 min, the supernatant containing CMD in excess was removed, and the pellet consisting of M-CMD was re-suspended in sterile water.

**Characterization of Magnetosomes at Various Stages of Synthesis, Including M-CMD:** The detailed description of the methods employed in Section 1.2 is provided is Supporting Information.

Transmission electron microscopy (TEM) – the sizes, distribution in sizes, shapes, crystallinity and organizations of the magnetosomes at the different steps of the fabrication process, i.e. magnetosomes contained in MSR-1 bacteria, pyrogenic chains of magnetosomes extracted from MTB, purified nonpyrogenic individual magnetosome minerals, and reconstructed chains of magnetosome minerals coated with carboxy-methyl-dextran (M-CMD), were measured or assessed using TEM (JEM-2100, JEOL, Japan).[53]

Magnetosome concentration measurements – the concentrations of M-CMD in suspension were measured by estimating the quantity of iron that they contain through a ferrous thiocyanate colorimetric assay using a spectrophotometer UviLine 9400, Secomam, France or an inductively coupled plasma mass spectrometry (ICP-MS) instrument (Agilent, 7900 ICP-MS).

Zeta Potential Measurements – Zeta potentials of nonpyrogenic magnetosomes (M) and M-CMD in suspension were measured as a function of the pH of these suspensions (2 < pH < 14) by Dynamic Light Scattering, using a ZEN 3600, Malvern Instruments, UK.

Stability of M-CMD in suspension – To determine if the M-CMD suspension was stable, the variation of its optical density was measured at 480 nm for 2 hours following the homogenization of these suspensions using a spectrophotometer (UviLine 9400). The details description of the method is provided in the Supporting Information.

Quantity of carbonaceous material contained in the various magnetosomes – The amount of organic material (carbon and nitrogen) contained in pyrogenic chains of magnetosomes, nonpyrogenic uncoated magnetosome minerals, and nonpyrogenic coated magnetosome minerals was estimated using an elemental CHNS analyzer (Flash EA 1112, Thermo Fisher Scientific, USA).

Quantity of endotoxins contained in the magnetosomes at various stages of synthesis – magnetosome pyrogenicity was evaluated by measuring the endotoxin concentration of the different samples using the Limulus amebocyte lysate (LAL) tests (32) (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Fisher Scientific, USA).

**In Vitro M-CMD Studies:** Culture of PC3 Cells – PC3 cells (CRL-1435, ATCC) were thawed in a water bath at 37°C for 5 min and introduced into a 75 cm²-culture flask containing 10 mL of DMEM without phenol red (GIBCO), supplemented with 10% v/v of FBS (GIBCO), 1% v/v of Penicillin-Streptomycin (GIBCO) and 1% v/v of HEPES buffer (GIBCO). After seeding in the flask, the cells were maintained at 37°C and 5% $CO_2$ in an incubator. After 3 passages of amplification cells, PC3 cells were ready for further experiments.

In vitro hyperthermia treatment using LIU – PC3 cells were seeded in sterile 6-well plates ($10^6$ cells/well) and incubated overnight at 37°C in a 5% $CO_2$ incubator. The medium was then removed and replaced with 1 mL of fresh medium containing (or not) 500 µg mL⁻¹ in iron of M-CMD and the plates were re-incubated for 3 h at 37°C at 5% $CO_2$. The supernatant of each well was collected in Eppendorf tubes of 2 mL and stored at room temperature. A volume of 1 mL of 0.25% Trypsin – EDTA was then added to each well to disperse the cell layer. The cells were then mixed with 1 mL of 100% FBS (Fetal Bovine Serum), harvested in 2 mL Eppendorf tubes and centrifuged at 1000 × g for 7 min. The solution of Trypsin/FBS was discarded, and the cells were resuspended with their corresponding supernatants in a total volume of 1 mL. Each Eppendorf tube filled with 1 mL of medium containing (or not) M-CMD was then dived upright up to the cap in a bath container (4 cm diameter) mounted a plane ultrasound transducer (3.6 cm diameter) and filled with degassed water. The cells were exposed to LIU of 1 W cm⁻² and 1 MHz for 10 or 20 min, or not exposed to LIU. This intensity level was chosen to be similar to that used in vivo to treat mouse tumors and to effectively reach the target heating temperature of 45–46°C.[54–59] During LIU treatment, the temperature was monitored over time using a flexible thermocouple probe (Physitemp, MT29-5HT) inserted into each tube and a DASYLab software used to plot temperature curves. To ensure that the temperature did neither exceed 46 °C nor fall below 44 °C during the treatment, the intensity of ultrasound was constantly adjusted.

Following treatment, the cells were re-seeded in new 6-well plates and incubated for 24 h at 37°C and 5% $CO_2$. Subsequently, the cell viability
was determined using resazurin reagent as described in Section 1.3.3., a propidium iodide/annexin V flow cytometry assay was employed to determine the type of PC3 cell death, as described in Section 1.3.4., and the degree of internalization of M-CMD into cells at different treatments was determined using ICP-MS, as detailed in Section 1.3.5.

CEM43 calculation – to evaluate the different times of hyperthermia treatment of PC3 cells by LIU, the method developed by Sapareto and Dewey[60] was used, which converts the time-temperature curve into an equivalent time at 43°C, designated CEM 43°C (cumulative equivalent minutes at 43°C). The calculation of CEM 43°C is expressed as follows

$$CEM43^{\circ}\text{C} = \sum_{i=1}^{n} ti \times R^{(43-Ti)} \quad (1)$$

where $ti$ is the time interval in minutes at a specific temperature, $Ti$ is the average temperature during the time interval $ti$, $R$ are the temperature-dependent rate of cell death constants either 0.25 when $T < 43^{\circ}\text{C}$ or 0.5 when $T > 43^{\circ}\text{C}$ and $n$ is the number of temperature intervals below or above the threshold temperature of 43°C. The same calculation was applied for the calculation of the thermal dose applied to the tumors in the in vivo experiments

Cell viability measurements – a volume of 110 µL of Alamar Blue was added to each 6-well plate (Section 1.3.2) and mixed under agitation at 120 rpm for 10 min. Subsequently, the plates were incubated for 3 hours at 37 °C in 5% CO₂ and the liquid was transferred to 1.5-mL Eppendorf tubes. The tubes were then centrifuged at 14100 × g for 10 min (Eppendorf, MiniSpin plus) to remove all nanoparticles which could interfere with the assay. Subsequently, 100 µL of the supernatant was transferred into a 96 well-plate to be measured at excitation and emission wavelengths of 530 and 590 nm, respectively using a fluorescence reader (LabSystems, Fluoroskan Ascent CF). The cell viability was calculated using the formula

$$Cell\ viability\ (%) = \left( \frac{Ft - Fb}{Fc - Fb} \right) \times 100\% \quad (2)$$

where $Ft$ is the fluorescence intensity of the supernatant of the wells containing the treated cells, $Fb$ is the fluorescence intensity of blank measured for empty wells, and $Fc$ is the fluorescence intensity of wells filled with 100 µL of cell culture medium mixed with 10 µL of resazurin.

Cellular death assay by flow cytometry measurement – the type of PC3 cellular death was determined 24 h after the various treatments (Section 1.3.2) using propidium iodide/annexin V flow cytometry for necrosis and apoptosis identification, respectively. The supernatant of each 6-well plate was transferred to tubes of 2 mL. The cells were detached by adding a volume of 1 mL of 0.25% Trypsin – EDTA, were resuspended with their corresponding supernatants in a volume of 1 mL and were filtered through a hydrophilic polycarbonate Isopore membrane (50 µm, 25 mm) (Merck) to remove extracellular magnetosomes. After that, each tube of 15 mL was centrifuged at 1000 × g for 7 min. The supernatants were removed and each pellet of cells was mixed with 500 µL of Buffer A which was prepared with 10.8 mL of WFI water and different solutions provided from Annexin V-FITC/PI Apoptosis Detection Kit (V13242, Invitrogen) (1.2 mL of binding buffer, 5 µL of propidium iodide (PI) and 5 µL annexin V-FITC). Each tube was incubated in the dark at room temperature for 30 min. Subsequently, 500 µL of PBS was added to each tube and mixed before flow cytometry analysis.

Stained cells were analyzed using a CytoFLEX (Beckman Coulter). Samples were excited using two lasers at 488 and 561 nm, and the fluorescence emitted by the sample was measured, both as forward scatter (FSC) and side scatter (SSC) signals which reflect the distribution in treated cell size and granulometry, respectively. Necrosis was assessed using PI staining after excitation at 561 nm and detection of the corresponding fluorescence signal using a 610/20 BP filter (ECD canal). Similarly, apoptosis was assessed using annexin V-FITC staining followed by excitation at 488 nm and fluorescence detection using a 525/40 BP filter (FITC canal).

Internalization of M-CMD into PC3 cells – the internalization of M-CMD into PC3 cells was determined 24 hours after the various treatments (Section 1.3.2) using ICP-MS. The supernatant of each 6-well plate was trans-

ferred to 15 mL tubes. The cells were resuspended with their corresponding supernatants in a total volume of 1 mL and were filtrated through a hydrophilic polycarbonate Isopore membrane (50 µm, 25 mm) (Merck) to remove extracellular magnetosomes. A volume of 10 µL was collected from each solution to determine the number of cells per tube using the Mallesez chamber. After that, each tube of 15 mL was centrifuged at 1000 × g for 7 min. The supernatants were removed and each pellet of cells was mixed with 100 µL of an aqua regia (HCl/HNO₃, w:v / w:v, 37%:3 / 70%:1) and incubated at 37 °C for 48 h for degradation. After that, 4 mL of WFI water were added to each tube and mixed before ICP-MS measurement in an inductively coupled plasma-mass spectrometer (Agilent, 7900 ICP-MS) to determine the intracellular iron content.

Statistical analysis – the data were analyzed using GraphPad Prism version 8.0.2. All measurements were performed in quintuplicate and data were reported as the mean ± standard deviation (SD). Statistical comparisons were carried out using one-way ANOVA and the differences were considered significant when *p* < 0.05, **p* < 0.01, and ***p* < 0.001. The alpha-value is 0.95.

*In Vivo Combined Ultrasound / M-CMD Treatments of PC3-Luc Tumors:* Ethics statement – in vivo experiments were carried out following the ethical guidelines of the Institutional Animal Care and Use Committee (“Ethics committee Charles Darwin N° 5”). In particular, mice were fed and received water according to these guidelines and were euthanized by cervical dislocation when their weight had decreased by more than 20% relative to their initial weight before tumor cell implantation, the tumor volume exceeded 1000 mm³ (from one measurement to another), or when signs of pain, unusual posture or prostration were observed. Nanobacterium received on 11/09/2018 authorization A751320 from the French ministry of research to lead a project entitled: “Targeted treatment of nonmetastatic prostate cancer in combination with several thermal treatments: magnetic hyperthermia, low frequency ultrasound, and laser” to carry out animal experiments described in this article.

Growth of subcutaneous PC3-Luc Xenografts in mice – PC3-Luc cells (Bioware Brite Cell Lines PC3-RedFLuc-GFP (ref BW133416) were cultivated and sub-cultured analogously as described in Section 1.3.1. for the PC3 cell line. After two weeks of acclimatization, 200 µL of a suspension containing 3.5 × 10⁷ PC3-Luc cells were injected subcutaneously with a needle syringe 26 G 1 mL (Terumo) under 2% isoflurane anesthesia (Iso-care) into the right flank of 152 pathogen-free 6-week-old athymic male nude mice of mean weight 20 g, purchased from Envigo, derived from the National Cancer Institute, Frederick, Maryland. Fifteen days after cancer cell injection, the tumors reached a volume between 80 and 150 mm³, with an average volume of 100 mm³, and the various treatments were started.

Treatment conditions of mice bearing subcutaneous PC3-Luc xenografts – the conditions of treatment of mice bearing subcutaneous PC3-Luc tumors and the experimental protocol used to treat mice are summarized in Table S1 and Figure S1 (Supporting Information), respectively. A total of 80 mice with implanted tumors, designated by MX where X is the mouse number, were randomly distributed into 8 different treatment groups. Treatment began at day 0 (D0). Then, the groups were variably injected intratumorally 30–50 µL of isotonic solutions (5% glucose) containing or not 10 mg mL⁻¹ of M-CMD at a dose of 3 µg of M-CMD per mm³ of tumor. In addition, the tumors were either delivered with 6 to 10 consecutive sessions (3 per week) of ultrasound starting at D0 up to D21 and numbered from S1 to S10 (S1 at D0, S2 at D1, S3 at D2, S4 at D7, S5 at D8, S6 at D9, S7 at D14, S8 at D15, S9 at D16 and S10 at D21), or not treated. Some animals received less than 10 ultrasound delivery sessions because their tumor had completely vanished as detected by palpation, and confirmed by BLI measurement (ultrasound treatment was discontinued as soon as no luminescence was assessable).

Group I (M1 to M10) was the nontreated control group, it received 5% glucose. Group II (M61 to M70) received 5% glucose and was exposed to ultrasound of 1 W cm⁻². The frequency of ultrasounds was always 1 MHz in all experiments. Group III (M11 to M20) received M-CMD. Group IV (M71 to M80) received M-CMD and was exposed to ultrasound of 1 W cm⁻². Group V (M51 to M60) received M-CMD and was exposed to ultrasound of 0.5 W cm⁻². Group VI (M31 to M40) received M-CMD and
was exposed to ultrasound of 0.2 W cm⁻². Group VII (M41 to M50) received 5% glucose and was exposed to ultrasound of 0.5 W cm⁻². Group VIII (M21 to M30) received 5% glucose and was exposed to ultrasound of 0.2 W cm⁻². All ultrasound applications (Figure S1, Supporting Information) were performed under isoflurane (2%) anesthesia using a Primo Therasonic460, Oxfordshire, UK device consisting of a Phyation ultrasound generator with a plane transducer with a diameter of 3.6 cm. A gel pad, a latex laboratory glove and an ultrasound gel (Winelec), were positioned between the transducer surface and the mouse skin to enable ultrasound transmission. Each ultrasound session lasted 10 minutes, divided between an initial period of temperature increase, and a second period of temperature stabilization at 43–46°C. For ultrasound of 1 W cm⁻² the distance between the transducer and the skin surface over the tumors was constantly adjusted manually during the sessions to maintain the tumor plateau temperature stable within a range of 43–45°C. This manual adjustment was not required to maintain plateau temperatures of 35–37°C for 0.2 W cm⁻² and 35–44°C for 0.5 W cm⁻², as the maximum temperature had been reached.

Temperature monitoring – during each ultrasound session in groups II, IV, V, VI, VII and VIII, intratumoral temperature was measured as a function of time locally at the site of glucose or M-CMD injection using a thermocouple with a microprobe (IT-18, Physitemp, Clifton, USA) placed in the tumor. The thermocouple was connected to the Dasy lab software, which showed and recorded in real-time the intratumoral temperature. Simultaneously, the rectal temperature was also measured to verify that the systemic body temperature was unaffected.

Bioluminescence imaging (BLI) – BLI was carried out once a week to follow tumor size evolution following the various treatments after D0 using an IVIS Spectrum System (in vivo imaging system, PerkinElmer, Inc., Walther, MA) for all groups (group I to VIII). To yield a BLI signal, a suspension of luciferin was administered intraperitoneally to the mice 10 minutes before BLI measurements. Under luciferin reaction with the ATP of living PC3-Luc cells, luminescent oxyluciferin is produced, hence resulting in a BLI signal, whose strength is proportional to the number of living PC3-Luc cells present in the detection region. The open-drop method of isoflurane exposure was used to anesthetize mice during the BLI measurement which was used for treated mice with nonpalpable/invisible tumors, for which BLI provides a means to verify the absence of any remains of tumor cells. Therefore, this method was used to guide the US treatment continuance.

Treatment efficacy assessed by tumor volume measurement, tumor length (L), width (W), and height (H) were measured by a caliper twice per week for all groups (group I to VIII) and the tumor volume was estimated using the following formula, which relates the tumor volume, V, to length, L, width, W, and height, H: $V = L \times W \times H$, assuming in first approximation a shape of a rectangular prism for the tumor. Mouse weight was measured with a dedicated scale at the same days as for tumor volume measurement to verify the health of the mice. Signs of pain, like prostration, reduction or lack of water drinking, were also monitored at these same days to determine any potential side effects induced by the treatment.

Statistical analysis – mouse survival rates were analyzed between D0 and D350 using the Kaplan-Meier model method and plotted between D0 and D60. The statistical significance of survival rates in the different groups was evaluated using the log-rank test. The parameters were expressed as the mean ± SD and as p-values estimated for groups II, III, IV, V, VI, VII and VIII relative to group I, and for groups IV, V and VI relative to group III, and for group II relative to group IV. The statistical significance of tumor volume differences in the different groups was evaluated using the Mann-Whitney test. The parameters were expressed as the mean ± SD and as p-values calculated for groups II, III, IV, V, VI, VII, VIII relative to group I, and for groups II, V, VI relative to group IV. The differences were considered significant when * *p* < 0.05, ** *p* < 0.01, and *** *p* < 0.001. The alpha-value is 0.95.

**Histological Analysis:** Preparation of the histological sections – histological analysis was carried out on 72 mice. The mice were randomly distributed into 8 groups according to the definitions of treatment groups I to VIII as described above. For each group 2 mice each were euthanized and sampled at 2h (D0), 24h (D1), 48h (D2) and 168h (D7) following the injection. In addition, two mice each from treatment group II and treatment

group IV were euthanized and sampled at 504h (D21) and 6 months following the injection. For all mice tissues from the engraftment site, were collected for histological analysis. For the mice euthanized at 6 months after injection, tissues from various organs (liver, spleen, lung, brain and kidney) were additionally collected.

After collection, tumor or organs were cut in halves longitudinally. All tissues were fixed in a solution of paraformaldehyde at 4% for 24–48h and dehydrated in graded ethanol and isopropanol (Logos One, Thermo Fisher Automaton) for 48–72h before being embedded into paraffin blocks. Sections of 4 µm thickness (Sakura Autosection) were mounted on Klinipath slides and stained as indicated below.

Perls staining – to highlight the presence, localization, distribution and internalization of iron originating from the magnetosomes, sections were deparaffinized, rinsed, and stained for 10 minutes in a solution containing 5% potassium ferrocyanide and HCl 1N, the nuclei were counterstained for 10 minutes by nuclear red (BioOptica). This staining was realized by an automaton (Spectra, Leica). Five stained slides per block were scanned using a Lamina slide scanner, Perkin Elmer and viewed using the Pannoramic viewer software 1.15.4.

H&E staining – H&E staining permitted to distinguish neoplastic from nonneoplastic areas, and also to evaluate the type and degree of damage caused to the tumor tissue by the treatments. For the standard morphological HE staining, sections were deparaffinized, rinsed, and stained 5 min in Mayer's hematoxylin (VWR international) and 8 minutes in 0.5% eosin Y (VWR international). This staining was realized by an automaton (Spectra, Leica). Five stained slides per block were scanned and viewed as indicated above. The extent of developing/complete necrosis was estimated visually as a percentage of the tumor area.

## Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

## Acknowledgements

The authors would like to thank the BPI ("banque publique d'investissement, France"), the region of Paris ("Paris Région Entreprise, Franc"), the French Research Tax Credit program ("crédit d'impôt recherche"), the incubator Paris Biotech Santé, the ANRT (CIFRE 2014/0359, CIFRE 2016/0747, CIFRE 2013/0364, CIFRE 2015/976), the Eurostars programs (Nanoneck-2 E9309, Nanoglioma E11778, Nanoprostate E114157), the AIR program ("aide à l'innovation responsable") from the region of Paris (A1401025Q), the ANR ("Agence Nationale de la Recherche") Méfisto, as well as the Universities Paris 6 and Paris 11. We also would like to thank the Nomis Foundation and Markus Reinhard for their support.

## Conflict of Interest

E. A., I. C., C. E. H. and R. L. F. have been employed by AlphaOnco (previously called Nanobacterie). E. A. is a co-founder of AlphaOnco and holds stocks in the company.

## Author Contributions

E.A. directed the research presented in this manuscript. C.E.H., I.C., R.L.F. carried out the experiments presented here. E.B., C.M., F.G., F.G., J.-F.A., O.S. participated in the set-up of various aspects of the methodologies involved in this study. All co-authors have red this manuscript and agreed about its content.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.
Keywords

hyperthermia, magnetosome, nanomedicine, prostate, ultrasound

Received: June 30, 2024

Revised: November 14, 2024

Published online: January 19, 2025

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