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Breast cancer is the most frequent cancer for women in western countries. Immunotherapy is used to make the anti-tumor immunity more efficient. Several studies showed that combination of radiotherapy and immunotherapy leads to a more efficient tumor rejection than when these two treatments are used separately.
The Pharmacotherapy lab showed that this synergy is conserved when the mammary tissue is irradiated 24h before tumor cell injection. This indicates that this synergy is at least partly due to the irradiation of the microenvironment rather than tumor cells themselves. Moreover, tumor eradication was accompanied by an increase of T CD8 lymphocyte infiltration compared to controls.
The aim of this work was to understand the mechanisms by which pre-irradiation of the mammary tissue improve tumor rejection by immunotherapy.
First, we tested the implication of several radio-induced cytokines like MIP-1 and IFN. We therefore overexpressed these cytokines in the mammary tissue by electrotransferring plasmids coding for those proteins. Our results indicate that overexpression of IFN improves T CD8 lymphocytes infiltration in tumors as soon as four days after vaccine administration while MIP-1 overexpression had no effect. However, the large variability of the electrotransfer technique renders the interpretation difficult. In parallel, we focused on the role of cell necrosis in our model because large necrotic regions were observed in tumors growing in pre irradiated environment. Injection of necrotic cells in vivo (by different ways) reveals a reduction in vascular density and an increase of lymphocyte infiltration in tumors of vaccinated mice.
Finally, we studied the role of macrophages in our model of immunotherapy. Macrophages are a major component of mammary tissue before tumor cell injection. First, we used clodronate liposomes to deplete macrophages in the mammary tissue. Tumors implanted in those tissues showed growth delay similar to the one observed with the pre-irradiated tissue. However, the effect of pre-irradiation of the mammary tissue, before tumor cell injection, was conserved in absence of macrophages. Secondly, we co-injected tumor cells with M1 or M2 macrophages in pre-irradiated tissue. The results of this experiment showed that the injection of M2 macrophages leads to a slower tumor regression.
In conclusion, our study showed that macrophages and necrosis are likely to be involved in the synergy observed between radio- and immunotherapy. Those results justify the interest for the tumor microenvironment in the search for new modalities to improve the immunotherapy efficiency Le cancer du sein est actuellement le cancer le plus fréquent chez la femme dans les pays occidentaux. L’immunothérapie a pour objectif de rendre la réponse immunitaire anti-tumorale efficace. Différentes études montrent que lorsqu’on associe un traitement par radiothérapie avec une immunothérapie, le rejet tumoral est plus efficace que lorsque ces traitements sont utilisés séparément.
Le laboratoire de Pharmacothérapie a montré que cette synergie était conservée lorsque le tissu mammaire était irradié 24h avant l’implantation de cellules tumorales au sein de celui-ci, indiquant un rôle du microenvironnement plutôt que de l’irradiation des cellules tumorales elles-mêmes. De plus, le rejet tumoral était accompagné d’une infiltration accrue de lymphocytes T CD8 par rapport à la vaccination seule.
L’objectif de ce travail, était de comprendre les mécanismes par lesquels la pré-irradiation du tissu mammaire améliore le rejet tumoral par immunothérapie.
Dans ce but, différentes pistes ont été suivies. Tout d’abord, nous nous sommes intéressés à l’implication de différentes cytokines dont l’expression est radio-induite, telles que MIP-1 et l’IFN. Pour cela, nous avons surexprimé ces cytokines dans le tissu mammaire en électrotransférant les plasmides codant pour ces protéines. Nos résultats montrent que la surexpression de l’IFN, semble augmenter l’infiltration des lymphocytes T CD8 dans les tumeurs dès 4 jours après la vaccination alors que la surexpression de MIP-1 est sans effet. La grande variabilité de la technique d’électrotransfert rend toutefois l’interprétation de ces résultats difficile. En parallèle, nous nous sommes intéressés au rôle de la nécrose dans notre modèle. En effet, de nombreuses plages de nécrose sont observées dans les tumeurs se développant dans un environnement irradié. Nous avons donc injecté des cellules nécrotiques in vivo par différentes voies. Ces expériences préliminaires ont montré une diminution de la vascularisation et une tendance à une augmentation de l’infiltration de lymphocytes T CD8 au sein des tumeurs dans les souris vaccinées.
Finalement, nous avons étudié le rôle des macrophages dans notre modèle d’immunothérapie. En effet, ces cellules sont présentes en grande quantité dans le tissu mammaire préalablement à l’injection de cellules tumorales. Nous avons, dans un premier temps, déplété les macrophages dans les tissus mammaires, à l’aide de liposomes contenant du clodronate. L’injection consécutive de cellules tumorales a donné lieu à une moindre croissance des tumeurs, similaire à celle observée lors de l’irradiation. L’effet de la pré-irradiation du tissu mammaire avant l’implantation de cellules tumorales est cependant conservé en absence de macrophages. Dans une seconde série de manipulations nous avons co-injecté des cellules tumorales avec des macrophages de sous-types M1 ou M2. Les résultats montrent que l’injection de cellules M2 avec les cellules tumorales mène à une régression plus lente des tumeurs.
A l’issue de ce travail, les macrophages semblent être impliqués dans la potentialisation de l’immunothérapie par la radiothérapie, mais ne peuvent pas expliquer à eux seuls les effets observés. La nécrose cellulaire semble également jouer un rôle dans cette synergie. Ces résultats justifient l’intérêt particulier porté au microenvironnement tumoral pour l’optimalisation de l’immunothérapie

Immunotherapy --- Radiotherapy --- Breast Neoplasms --- Macrophages --- Necrosis

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In light of the critical contributions of macrophages and dendritic cells to diverse inflammatory diseases and to immunity and host defense, state-of-the-art approaches to the investigation of their behavior are essential. In Macrophages and Dendritic Cells: Methods and Protocols, expert researchers contribute laboratory protocols involving these two vital cell types functioning at the junction of the innate and acquired immune systems. The volume delves first into isolation and cell culturing then continues with topics such as phagocytosis, genetic manipulation, macrophage activation, and lipid signaling. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Macrophages and Dendritic Cells: Methods and Protocols provides a timely and useful guide for both seasoned investigators and neophytes pursuing this imperative field of study.

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Tumor necrosis factor (TNF) superfamily is a rapidly growing family of cytokines that interacts with a corresponding superfamily of receptors. Ligand-receptor interactions of this superfamily are involved in numerous biological processes ranging from hematopoiesis to pleiotropic cellular responses, including activation, proliferation, differentiation, and apoptosis. The particular response depends on the receptor the cell type, and the concurrent signals received by the cell. Worldwide interest in the TNF field surged dramatically early in 1984 with the cloning and defining of the profound cellular effects of the first member of this family, TNFa. Subsequently, the major influence of TNFa on the development and functioning of the immune system was established. Today, over 20 human TNF ligands and their more than 30 corresponding receptors have been identified. Few receptors still remain orphans. What has emerged over the years is that most TNF ligands bind to one distinct receptor and some of the TNF ligands are able to bind to multiple TNF receptors, explaining to some extent the apparent disparity in the number of TNF receptors and ligands. Yet, in spite of some redundancy in TNF ligand/receptor interactions, it is clear that in vivo spatial, temporal, and indeed cell- and tissue-specific expression of both ligands and their receptors are important factors in determining the precise nature of cellular physiological and pathological processes they control. Therapeutic Targets of the TNF Superfamily presents the state-of-the art account on the role of TNF superfamily members in the pathogenesis and their use in current intervention of cancers and autoimmune disease. This text will be highly valuable for investigators to understand the disease processes regulated by TNF superfamily members and to develop effective therapeutics. A view into the future, inspired by the comprehensive work presented in this volume, predicts that researchers studying TNF superfamily members will continue to make rapid progress in identifying relevant components to the disease process and new therapeutic strategies to target many human diseases including cancers, autoimmune disease and others.

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