Anticancer chemotherapy and radiotherapy trigger both non-cell-autonomous and cell-autonomous death.

Even though cell death modalities elicited by anticancer chemotherapy and radiotherapy have been extensively studied, the ability of anticancer treatments to induce non-cell-autonomous death has never been investigated. By means of multispectral imaging flow-cytometry-based technology, we analyzed the lethal fate of cancer cells that were treated with conventional anticancer agents and co-cultured with untreated cells, observing that anticancer agents can simultaneously trigger cell-autonomous and non-cell-autonomous death in treated and untreated cells. After ionizing radiation, oxaliplatin, or cisplatin treatment, fractions of treated cancer cell populations were eliminated through cell-autonomous death mechanisms, while other fractions of the treated cancer cells engulfed and killed neighboring cells through non-cell-autonomous processes, including cellular cannibalism. Under conditions of treatment with paclitaxel, non-cell-autonomous and cell-autonomous death were both detected in the treated cell population, while untreated neighboring cells exhibited features of apoptotic demise. The transcriptional activity of p53 tumor-suppressor protein contributed to the execution of cell-autonomous death, yet failed to affect the non-cell-autonomous death by cannibalism for the majority of tested anticancer agents, indicating that the induction of non-cell-autonomous death can occur under conditions in which cell-autonomous death was impaired. Altogether, these results reveal that chemotherapy and radiotherapy can induce both non-cell-autonomous and cell-autonomous death of cancer cells, highlighting the heterogeneity of cell death responses to anticancer treatments and the unsuspected potential contribution of non-cell-autonomous death to the global effects of anticancer treatment

Kinetic study of an immune response associated with tumor regression : T lymphocytes and myeloid cells cooperate within the tumor after vaccination

De nombreuses études en oncoimmunologie portent sur l’échec immunitaire dans le contexte de progression tumorale, mais elles sont plus rares à porter sur un contexte de régression, lorsque le système immunitaire est efficace. De ce fait, bien souvent la littérature met en avant le rôle cytotoxique des lymphocytes T CD8+, ou bien leur anergie dans le contexte de progression tumorale, causée par des cellules myéloïdes telles que les MDSC ou les macrophages de phénotypes M2, considérés comme pro-tumoraux. J’ai pour ma part étudié la réponse immunitaire dans le cadre d’une régression tumorale. Des cellules TC1 transplantées en s.c. dans des souris C57BL6/J, donnent des tumeurs solides d’environ 6mm de diamètre 11 jours plus tard. A ce moment là (J0), les souris sont vaccinées à proximité de la tumeur (priming), par un vaccin contenant la sous-unité B non toxique de la Shiga toxine couplée au peptide E7 de l’HPV16 (exprimé par les TC1), combiné à de l’IFNα. Une semaine plus tard (J7), un « boost » est effectué. Après le boost, la croissance tumorale cesse puis la tumeur régresse. L’analyse cinétique par cytométrie révèle un infiltrat immunitaire important pendant, et précédant la régression tumorale. La nature de cette infiltrat varie avec le temps. A J5, un infiltrat myéloïde est observé, suivi d’un infiltrat lymphocytaire à partir de J8. Une déplétion des cellules T CD8+ inhibe la régression tumorale, alors que dans les souris CXCR3-/-, dans lesquelles les CD8+ ne sont pas déplétés mais leur recrutement est fortement affecté, une régréssion tumorale est possible malgré un infiltrat T CD8+ très faible. Cela laisse penser que d’autres acteurs que les LT cytotoxiques sont nécessaires à la régression tumorale, comme probablement les cellules myéloïdes qui infiltrent le tumeur avant les cellules T. L’analyse de cette population montre une activation des monocytes et macrophages (MHC II+), avec un pic d’activation autour de J9, au début de la régression. La capacité cytotoxique de ces cellules, mesurée in vitro par immunofluorescence est augmentée comparée à des myéloïdes isolées de tumeurs de souris en progression. De plus, l’ajout d’un anticorps anti-TNFα inhibe partiellement cette cytotoxicité. Cela montre qu’après vaccination, les monocytes/macrophages sont capables de tuer les cellules tumorales. Une déplétion partielle des macrophages au moment de la vaccination, à l’aide du PLX3397 (inhibiteur du CSF1R), réduit l'efficacité de la vaccination. Les cellules myéloïdes, lorsqu'elles sont présentes, contribuent fortement à la régression tumorale induit par le vaccin composite, et leur action implique probablement des interactions avec les LT CD8+. C'est ce que suggère l'observation de tumeurs vaccinées dans des souris IFNϒ-/-, dans lesquelles l'efficacité vaccinale est aussi inhibée. Cette thèse montre qu’après une stimulation appropriée, qui peut, comme ici, mimer une infection virale, les cellules myéloïdes peuvent participer activement à la régression tumorale.Most oncoimmunology studies are performed in an immune failure context of progressing tumor. They rarely describe tumor regressions, when the immune response is efficient. As a result, the literature tends to highlight the cytotoxic role of CD8+ T cell or their anergy in the context of tumor progression, caused by myeloid cells such as the MDSC or M2 polarized macrophages, considered as protumoral. My PhD work has been focused on the immune response in a context of tumor regression. TC1 cells transplanted s.c. in C57 BL6 J mice, give rise to solid tumors of approximately 6 mm diameter 11 days later. At that time (day 0), mice are vaccinated peritumorally for a priming with a composite vaccine containing the subunit B of the Shiga toxin coupled to E7 peptide from HPV16 (present on TC1), combined with the IFNα. A week later (day 7), a boost is made. After the boost, tumor growth stops and the tumor regress. Kinetic cytometric analysis revealed a significant immune infiltrate during and prior to tumor regression. The nature of this infiltrate varies with time. On day 5, a myeloid infiltrate is observed, followed by a lymphocytic infiltrate which is conspicuous after day 8. Depletion of CD8+ T cells inhibits tumor regression, while in CXCR3- /- mice, in which the CD8+ are not depleted but their recruitment is severely affected, tumor regression is possible despite a very low CD8+ T cell infiltrate. This suggests that some effectors, other than cytotoxic T cells, are required for tumor regression, including probably myeloid cells that infiltrate the tumor before T cells. The analysis of this population shows an activation of monocytes and macrophages (MHC II+) with a peak of activation around day 9, early in the regression. The cytotoxic capacity of these cells was tested in vitro, by depositing F4/80+ cells from vaccinated tumors or not, on a TC1 cell monolayers in culture. Only myeloid cells from vaccinated tumors appear to kill tumor cells, and adding an anti-TNFα inhibits this cytotoxicity. This shows that after immunization, monocytes/macrophages are capable of killing tumor cells. A partial depletion of macrophages at the time of vaccination, after treatment with PLX3397 (CSF1R inhibitor), reduces the vaccine efficacy. Myeloid cells contribute significantly to the observed tumor regression, and their action involves interactions with CD8+ T cells. This hypothesis is consistent with the observation of tumors in vaccinated IFNϒ- /- mice, in which the vaccine efficacy is also inhibited. This thesis shows that after an appropriate stimulation, for instance, here, by mimicking a viral infection, myeloid cells can actively participate in tumor regression

Etude cinétique d’une réponse immune associée à une régression tumorale : les lymphocytes T et les cellules myéloïdes coopèrent au sein de la tumeur après vaccination

Most oncoimmunology studies are performed in an immune failure context of progressing tumor. They rarely describe tumor regressions, when the immune response is efficient. As a result, the literature tends to highlight the cytotoxic role of CD8+ T cell or their anergy in the context of tumor progression, caused by myeloid cells such as the MDSC or M2 polarized macrophages, considered as protumoral. My PhD work has been focused on the immune response in a context of tumor regression. TC1 cells transplanted s.c. in C57 BL6 J mice, give rise to solid tumors of approximately 6 mm diameter 11 days later. At that time (day 0), mice are vaccinated peritumorally for a priming with a composite vaccine containing the subunit B of the Shiga toxin coupled to E7 peptide from HPV16 (present on TC1), combined with the IFNα. A week later (day 7), a boost is made. After the boost, tumor growth stops and the tumor regress. Kinetic cytometric analysis revealed a significant immune infiltrate during and prior to tumor regression. The nature of this infiltrate varies with time. On day 5, a myeloid infiltrate is observed, followed by a lymphocytic infiltrate which is conspicuous after day 8. Depletion of CD8+ T cells inhibits tumor regression, while in CXCR3- /- mice, in which the CD8+ are not depleted but their recruitment is severely affected, tumor regression is possible despite a very low CD8+ T cell infiltrate. This suggests that some effectors, other than cytotoxic T cells, are required for tumor regression, including probably myeloid cells that infiltrate the tumor before T cells. The analysis of this population shows an activation of monocytes and macrophages (MHC II+) with a peak of activation around day 9, early in the regression. The cytotoxic capacity of these cells was tested in vitro, by depositing F4/80+ cells from vaccinated tumors or not, on a TC1 cell monolayers in culture. Only myeloid cells from vaccinated tumors appear to kill tumor cells, and adding an anti-TNFα inhibits this cytotoxicity. This shows that after immunization, monocytes/macrophages are capable of killing tumor cells. A partial depletion of macrophages at the time of vaccination, after treatment with PLX3397 (CSF1R inhibitor), reduces the vaccine efficacy. Myeloid cells contribute significantly to the observed tumor regression, and their action involves interactions with CD8+ T cells. This hypothesis is consistent with the observation of tumors in vaccinated IFNϒ- /- mice, in which the vaccine efficacy is also inhibited. This thesis shows that after an appropriate stimulation, for instance, here, by mimicking a viral infection, myeloid cells can actively participate in tumor regression.De nombreuses études en oncoimmunologie portent sur l’échec immunitaire dans le contexte de progression tumorale, mais elles sont plus rares à porter sur un contexte de régression, lorsque le système immunitaire est efficace. De ce fait, bien souvent la littérature met en avant le rôle cytotoxique des lymphocytes T CD8+, ou bien leur anergie dans le contexte de progression tumorale, causée par des cellules myéloïdes telles que les MDSC ou les macrophages de phénotypes M2, considérés comme pro-tumoraux. J’ai pour ma part étudié la réponse immunitaire dans le cadre d’une régression tumorale. Des cellules TC1 transplantées en s.c. dans des souris C57BL6/J, donnent des tumeurs solides d’environ 6mm de diamètre 11 jours plus tard. A ce moment là (J0), les souris sont vaccinées à proximité de la tumeur (priming), par un vaccin contenant la sous-unité B non toxique de la Shiga toxine couplée au peptide E7 de l’HPV16 (exprimé par les TC1), combiné à de l’IFNα. Une semaine plus tard (J7), un « boost » est effectué. Après le boost, la croissance tumorale cesse puis la tumeur régresse. L’analyse cinétique par cytométrie révèle un infiltrat immunitaire important pendant, et précédant la régression tumorale. La nature de cette infiltrat varie avec le temps. A J5, un infiltrat myéloïde est observé, suivi d’un infiltrat lymphocytaire à partir de J8. Une déplétion des cellules T CD8+ inhibe la régression tumorale, alors que dans les souris CXCR3-/-, dans lesquelles les CD8+ ne sont pas déplétés mais leur recrutement est fortement affecté, une régréssion tumorale est possible malgré un infiltrat T CD8+ très faible. Cela laisse penser que d’autres acteurs que les LT cytotoxiques sont nécessaires à la régression tumorale, comme probablement les cellules myéloïdes qui infiltrent le tumeur avant les cellules T. L’analyse de cette population montre une activation des monocytes et macrophages (MHC II+), avec un pic d’activation autour de J9, au début de la régression. La capacité cytotoxique de ces cellules, mesurée in vitro par immunofluorescence est augmentée comparée à des myéloïdes isolées de tumeurs de souris en progression. De plus, l’ajout d’un anticorps anti-TNFα inhibe partiellement cette cytotoxicité. Cela montre qu’après vaccination, les monocytes/macrophages sont capables de tuer les cellules tumorales. Une déplétion partielle des macrophages au moment de la vaccination, à l’aide du PLX3397 (inhibiteur du CSF1R), réduit l'efficacité de la vaccination. Les cellules myéloïdes, lorsqu'elles sont présentes, contribuent fortement à la régression tumorale induit par le vaccin composite, et leur action implique probablement des interactions avec les LT CD8+. C'est ce que suggère l'observation de tumeurs vaccinées dans des souris IFNϒ-/-, dans lesquelles l'efficacité vaccinale est aussi inhibée. Cette thèse montre qu’après une stimulation appropriée, qui peut, comme ici, mimer une infection virale, les cellules myéloïdes peuvent participer activement à la régression tumorale

Design of RGD–ATWLPPR peptide conjugates for the dual targeting of α V β 3 integrin and neuropilin-1

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Nanoprobe Synthesized by Magnetotactic Bacteria, Detecting Fluorescence Variations under Dissociation of Rhodamine B from Magnetosomes following Temperature, pH Changes, or the Application of Radiation

International audienceWe report a method of fabrication of fluorescent magnetosomes, designated as MCR400, in which 400 μM of rhodamine B are introduced in the growth medium of AMB-1 magnetotactic bacteria and fluorescent magnetosomes are then extracted from these bacteria. These fluorescent magnetosomes behave differently from most fluorescent nanoprobes, which often lead to fluorescence losses over time due to photobleaching. Indeed, when MCR400 are heated to 30–90 °C, brought to an acidic pH, or exposed to radiations, we observed that their fluorescence intensity increased. We attributed this behavior to the dissociation of rhodamine B from the magnetosomes. Interestingly, enhanced fluorescence was also observed in vitro when MCR400 were mixed with either primary macrophages or tumor cells (TC1-GFP or RG2-Cells) or in vivo when MCR400 were introduced in rat glioblastoma. We showed that MCR400 internalize in tumor and immune cells (macrophages) leading to enhanced fluorescence, suggesting that fluorescent magnetosomes could be used during cancer treatments such as magnetic hyperthermia to image cells of interest such as immune or tumor cells

A Fluorescent Nanoprobe for the Detection of in Situ Temperature Changes during Hyperthermia Treatment of Tumors

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A Fluorescent Nanoprobe for the Detection of in Situ Temperature Changes during Hyperthermia Treatment of Tumors

Poster présenté lors d'une conférence en février 2018 à San Francisco. 62nd Annual Meeting Biophysical society 17/02/2018-21/02/2018International audienceMagnetic hyperthermia is a promising new treatment, allowing to locally induce a temperature increase in cancer tumors that leads to a lethal effect. For this, magnetic nanoparticles are introduced in tumors and exposed to an alternative magnetic field which produces the wanted temperature rise. While the final biological effect can be assessed by many techniques, the in situ temperature changes are often difficult to evaluate otherwise than with a regular thermometer. This fairly crude procedure does not allow to finely report changes at the tissue or cell level. In this context, we report here an original method based on a chemical nanoprobe designed to follow temperatures changes during hyperthermia therapy. In our work, AMB-1 magnetotactic bacteria produce the magnetic nanoparticles (magnetosomes), since we have already shown that this type of nanoparticles had a much better magnetic activity than chemically synthesized particles (Alphandery et al. ACS Nano, 2011, 5:6279). Interestingly, by introducing rhodamine B in an optimized growth medium for these bacteria, we were able to extract fluorescent magnetosomes with new characteristics. Indeed, keeping their typical magnetic activity useful for cancer therapy, they would also display a temperature-dependence fluorescence allowing to perform local measurements at a microscopic level in biological tissues. The molecular mechanism would be discussed, as well as results obtained with different cell types (RG2, TC1-GFP, C57NL/6 peritoneal macrophages, U87-MG) and tissues (RG2-implanted rat brain)

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse

International audienceBackground Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. Methods We designed a mouse model of tumor relapse in which mice transplanted with E7 + TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. Results We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. Conclusions Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse

“Polymultivalent” Polymer–Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing α v β 3 Integrins

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