Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy

Abstract

Purpose

Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts.

Materials and methods

B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice.

Results

In vitro analysis of bystander cytotoxic effects showed that CM killed about 30–40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects.

Conclusions

This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

Keywords:

• Targeted radionuclide therapy
• radiotherapy
• Auger electrons
• exosomes
• extracellular vesicles

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This work was supported by, SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, French National Research Agency as part of the Investissements d'Avenir program (ANR-10-LABX-53-01) and Cancéropôle Grand Sud Ouest, fondation ARC pour la recherche sur le cancer.

Notes on contributors

Jihad Karam

Jihad Karam, MSc, is a final year PhD student in Pr. J-P. Pouget's Laboratory at IRCM/INSERM Montpellier, France.

Julie Constanzo

Julie Constanzo is Research Scientist in Pr. J-P. Pouget's Laboratory at IRCM/INSERM Montpellier, France. She obtained her PhD in Physics and Radiobiology in 2013 (University Lyon1, France). Dr. Constanzo research project focuses on non-targeted effects caused by targeted radionuclide therapy including both intercellular communications and the immune response.

Alexandre Pichard

Alexandre Pichard has a Ph.D in radiobiology from the French National Institute for Health and Medical research (INSERM), Institut de Recherche en Cancérologie de Montpellier (U1194), Montpellier, France. His research interests are radiobiology and particularly the development of new therapies using the ionising radiations to treat cancer.

Laurent Gros

Laurent Gros is reseacrh scientist at IRCM/INSERM Montpellier, France, in the Immunity and cancer team led by Dr N Bonnefoy. He works in the field of immunology.

Joël Chopineau

Joël Chopineau is Professor of Biochemistry at Ecole Nationale Superieure de Chimie Montpellier. Institute for Molecular Chemistry and Material Sciences.

Ecole Normale Supérieure Paris Saclay, 1984 and Ph.D, 1989. His research concerns the design and development of membrane models and platforms dedicated to protein and nanoparticles–membrane interactions.

Marie Morille

Marie Morille is associate professor in Montpellier University (Pharmacy, Pharmaceutics development and biomaterials Department) and Institute Charles Gerhardt of Montpellier since 2012. Her research mainly focused on encapsulation/vectorization of biomolecules (nucleic acids, proteins) using micro-nanocarriers (synthetic and natural) for health applications (cancerology, tissue engineering) with a special care on coordination between carriers physico-chemical properties and impact on cell behavior.

Jean-Pierre Pouget

Jean-Pierre Pouget is INSERM Research Director and Team leader at IRCM/INSERM Montpellier, France. He obtained his PhD in 2000 from Curie Institute. He now works on the radiobiology of targeted radionuclide therapy with a focus on the contribution of non-targeted (bystander and immune) effects.