Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA^®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3⁺, CD4⁺, CD8⁺ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell’s cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery.

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary approach in immunotherapy that has shown remarkable success in the treatment of blood cancers. Many preclinical studies are currently underway worldwide to extend the CAR-T-cell therapy benefits to a broad spectrum of cancers, using rodent models. Alternative in vivo platforms are essential for overcoming the drawbacks associated with rodent models, including immunodeficiency in humanized models, ethical concerns, extended time requirements, and cost. In this work, we used the chicken chorioallantoic membrane (CAM) assay to evaluate the in vivo efficacy of cluster-of-differentiation 19 (CD19)-targeting CAR-T cells expressing a second-generation CAR construct against human lymphoma derived from the Raji cell line. Our results confirm the efficacy of selected CAR-T cells on tumor growth, metastasis, and angiogenesis. Further, the chicken embryo has an intrinsic active immune system. Therefore, the dialog between CAR-T cells and endogenous immune cells, as well as their participation in the tumor challenge, has also been studied. In conclusion, our study demonstrates that the chicken CAM assay provides a relevant in vivo, 3Rs (Replacement, Reduction and Refinement)-compliant new approach methodology (NAM), which is well-suited for the current needs of preclinical research on CAR-T-cell therapy.

Le cancer du sein était au premier rang des cancers chez la femme en termes d'incidence et de mortalité en 2008 (GLOBOCAN 2012, IARC). A l'origine de cette mortalité on observe notamment le développement de résistances aux chimiothérapies conventionnelles mais aussi le développement des métastases qui sont responsables de 90% des décès des patientes. Il est donc indispensable d'étendre l'arsenal thérapeutique à la disposition des cliniciens avec de nouvelles molécules actives sur les formes de cancers résistants aux chimiothérapies et ciblant le processus métastatique.Au sein de notre équipe nous avons identifié un nouvel inhibiteur de la LIM Kinase (LIMK) baptisé « Pyr1 ». La LIMK est responsable de la régulation de la dynamique des microtubules et des microfilaments d'actine. Dans un premier article (Prudent et al., Cancer Research, 2012) nous avons montré que Pyr1 avait une activité anti-mitotique et anti-migratoire dans un modèle cellulaire de cancer de l'utérus. De plus une analyse pilote a montré que Pyr1 était efficace dans un modèle murin de leucémie où il augmente la survie des souris. Étant donné que la LIMK est surexprimée dans les cancers du sein et qu'une des conséquences cellulaires d'un traitement par Pyr1 est une stabilisation des microtubules, comme le fait le paclitaxel couramment utilisé pour le traitement des formes invasives de ces cancers , l'objectif de ma thèse a été d'étudier l'efficacité thérapeutique de Pyr1 sur des modèles cellulaires et murins de cancer du sein, et notamment sur les cancers résistants au paclitaxel.Nous avons montré que Pyr1 ralentissait la croissance des tumeurs primaires et réduisait leur taille. Pyr1 est bien toléré et l'effet anti-tumoral est également observé sur des modèles résistants au paclitaxel. Nous avons ensuite étudié l'effet de Pyr1 sur la migration et l'invasion des cellules tumorales en utilisant notamment la microscopie intra-vitale. Nous avons observé que, bien qu'il ne diminue pas la vitesse de migration des cellules tumorales, Pyr1 entraine in vivo un changement morphologique important. Enfin, Pyr1 n'empêche pas la dispersion métastatique mais il prévient de manière efficace la croissance des métastases.Nous en avons conclu que Pyr1 est une molécule intéressante pour le traitement des cancers du sein résistants aux chimiothérapies conventionnelles et également sur le traitement des métastases.

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis and patient death. Dormancy is a non-proliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and proliferative D2A1 breast cancer cell line models in vivo and in vitro , and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro , we show that D2.OR cells are polyploid ER ⁺ /Her2 ⁺ cells, and in response to a 3D environment are growth arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible, and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.