Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma.

International audience: BackgroundIt is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse.ResultsUsing an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal.ConclusionsWe conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor

Facteurs inflammatoires et contrôle de la quiescence/activation des cellules souches tumorales de mélanome

Accumulating data suggest that both cancer development and recurrence depend on the ability of resistant tumor cells to adopt a quiescent or dormant phenotype following treatment. These dormant cells reside in various tissues of patients in complete remission without any clinical manifestation until they reactivate and cause tumor recurrence. Mechanisms that control the activation of quiescent tumor cells remain poorly understood, however, the tumor microenvironment, cellular interactions and various diffusible factors appear essential. Herein, our goal is to decipher whether a major pro-inflammatory cytokine, Tumor Necrosis Factor (TNF) contributes to the quiescence/activation phenotypic switch in melanoma. For this purpose, we used a 3D melanosphere and the in vivo-like skin equivalent models in which to reconstitute the in vivo-relevant cellular heterogeneity and tumor organization and an inducible histone 2B coupled to the GFP (H2B-GFP) expression system to identify the quiescent cell compartment and to monitor the TNF-induced changes. Our results suggest that TNF increases the proportion of H2B-GFP-positive, label retaining cells (LRC) in melanospheres. The LRCs were enriched in melanoma stem cell markers, ABCB5 and VEGFR and this was upregulated by TNF. Furthermore, TNF increases the number of melanospheres, and in skin equivalents, the presence of TNF seems to inhibit the differentiation of melanoma cells and increase the stem cell compartment. This effect appears to be governed by the activation of the PI3K / Akt pathway. In conclusion, these data show that inflammatory environment induced by TNF, activates melanoma quiescent stem cells and increases the compartment of stem cells in skin equivalents by preventing their differentiation. Therefore, the control of inflammation and signaling pathways involved in the maintenance of tumor dormancy during the treatment of the original tumor would be a good therapeutic strategy in the fight against cancer recurrences.A lot of data suggest that a cancer cell subpopulation is able to enter quiescence in response to cancer therapy, therefore we have also studied the effects of the first targeted therapy of melanoma: vemurafenib, on the stem cell compartment. Our results show that vemurafenib increases the number of melanospheres and the percentage of ABCB5+ cells. So vemurafenib increases the melanoma stem cell compartment. Vemurafenib increases also the percentage of H2B-GFP + cells and the percentage of cells in the GO phase of the cell cycle, so induces quiescence of melanoma cells. We also showed that vemurafenib stimulates activation of proteins regulating quiescence of stem cells.We hope that our research will provide new knowledge about the mechanisms that control the activation of quiescent cancer stem cells and provide new perspectives for the treatment of cancer.Une tumeur est composée de plusieurs sous populations cellulaires. L’une d’entre elles, celle des cellules souches tumorales, est à l’origine du développement des tumeurs. Une des propriétés majeures de ces cellules est la capacité d’entrer dans un état de quiescence. De ce fait, elles sont résistantes aux thérapies anticancéreuses conventionnelles qui visent les cellules cyclantes et peuvent ainsi persister pendant de nombreuses années. Ce phénomène est appelé dormance tumorale. L’activation de ces cellules souches tumorales quiescentes conduit à la récidive de la maladie. Le passage de l’état quiescent à l’état activé serait réversible, cependant les mécanismes responsables ne sont pas encore connus. Notre hypothèse est que les facteurs inflammatoires stimulent la transition des cellules de l’état quiescent à l’état activé. Dans ce but, nous avons étudié les effets de la principale cytokine pro-inflammatoire, le TNF, sur le compartiment des cellules souches de mélanome et leur activation. Pour cela, nous avons utilisé un système d’expression, inductible par la tétracycline, qui nous a permis d’identifier et d’étudier les cellules quiescentes H2B-GFP positives et cela dans les modèles in vitro des mélanosphères et des équivalents de peaux humaines reconstruites, afin de se rapprocher de l’organisation tumorale in vivo. Grâce à des tests fonctionnels, comme la formation de mélanosphères et de colonies, et diverses techniques telles que la cytométrie en flux, la microscopie à fluorescence et l’analyse de l’expression de gènes au niveau protéique, nous avons mis en évidence que les cellules H2B-GFP positives (« label retaining cells ») au sein des mélanosphères montrent un enrichissement en marqueurs de cellules souches du mélanome (ABCB5, VEGFR). De plus, nous avons montré que le TNF agit sur le compartiment des cellules souches. En effet, un traitement au TNF augmente le pourcentage de cellules exprimant des marqueurs de cellules souches de mélanome, inhibe la différenciation des cellules de mélanome (inhibition de l’expression de Melan-A dans les mélanosphères et diminution de la pigmentation des équivalents de peau), active les cellules souches quiescentes et induit des effets qui perdurent après le retrait du TNF. Notre étude a montré que ces effets seraient causés par une activation des voies PI3K/Akt et NFκB par le TNF. Un grand nombre de données suggérant qu’une sous-population de cellules cancéreuses est capable d’entrer en quiescence en réponse à une thérapie anticancéreuse, nous avons également étudié les effets de la première thérapie ciblée du mélanome : le vemurafenib, sur le compartiment des cellules souches. Nos résultats ont montré que le vemurafenib augmente le compartiment des cellules souches de mélanome (augmentation du nombre de mélanosphères formées et du pourcentage de cellules exprimant un marqueur de cellules souches de mélanome : ABCB5) et induit leur quiescence (augmentation du pourcentage de cellules H2B-GFP+ et en phase GO du cycle cellulaire). Nous avons également montré que le vemurafenib stimule l’activation de protéines régulant la quiescence des cellules souches.Nous espérons que nos recherches apporteront de nouvelles connaissances sur les mécanismes qui contrôlent l’activation des cellules souches cancéreuses quiescentes et offrir de nouvelles perspectives pour le traitement du cancer

Inflammatory factors and control of quiescence / activation of melanoma cancer stem cell

Une tumeur est composée de plusieurs sous populations cellulaires. L’une d’entre elles, celle des cellules souches tumorales, est à l’origine du développement des tumeurs. Une des propriétés majeures de ces cellules est la capacité d’entrer dans un état de quiescence. De ce fait, elles sont résistantes aux thérapies anticancéreuses conventionnelles qui visent les cellules cyclantes et peuvent ainsi persister pendant de nombreuses années. Ce phénomène est appelé dormance tumorale. L’activation de ces cellules souches tumorales quiescentes conduit à la récidive de la maladie. Le passage de l’état quiescent à l’état activé serait réversible, cependant les mécanismes responsables ne sont pas encore connus. Notre hypothèse est que les facteurs inflammatoires stimulent la transition des cellules de l’état quiescent à l’état activé. Dans ce but, nous avons étudié les effets de la principale cytokine pro-inflammatoire, le TNF, sur le compartiment des cellules souches de mélanome et leur activation. Pour cela, nous avons utilisé un système d’expression, inductible par la tétracycline, qui nous a permis d’identifier et d’étudier les cellules quiescentes H2B-GFP positives et cela dans les modèles in vitro des mélanosphères et des équivalents de peaux humaines reconstruites, afin de se rapprocher de l’organisation tumorale in vivo. Grâce à des tests fonctionnels, comme la formation de mélanosphères et de colonies, et diverses techniques telles que la cytométrie en flux, la microscopie à fluorescence et l’analyse de l’expression de gènes au niveau protéique, nous avons mis en évidence que les cellules H2B-GFP positives (« label retaining cells ») au sein des mélanosphères montrent un enrichissement en marqueurs de cellules souches du mélanome (ABCB5, VEGFR). De plus, nous avons montré que le TNF agit sur le compartiment des cellules souches. En effet, un traitement au TNF augmente le pourcentage de cellules exprimant des marqueurs de cellules souches de mélanome, inhibe la différenciation des cellules de mélanome (inhibition de l’expression de Melan-A dans les mélanosphères et diminution de la pigmentation des équivalents de peau), active les cellules souches quiescentes et induit des effets qui perdurent après le retrait du TNF. Notre étude a montré que ces effets seraient causés par une activation des voies PI3K/Akt et NFκB par le TNF. Un grand nombre de données suggérant qu’une sous-population de cellules cancéreuses est capable d’entrer en quiescence en réponse à une thérapie anticancéreuse, nous avons également étudié les effets de la première thérapie ciblée du mélanome : le vemurafenib, sur le compartiment des cellules souches. Nos résultats ont montré que le vemurafenib augmente le compartiment des cellules souches de mélanome (augmentation du nombre de mélanosphères formées et du pourcentage de cellules exprimant un marqueur de cellules souches de mélanome : ABCB5) et induit leur quiescence (augmentation du pourcentage de cellules H2B-GFP+ et en phase GO du cycle cellulaire). Nous avons également montré que le vemurafenib stimule l’activation de protéines régulant la quiescence des cellules souches.Nous espérons que nos recherches apporteront de nouvelles connaissances sur les mécanismes qui contrôlent l’activation des cellules souches cancéreuses quiescentes et offrir de nouvelles perspectives pour le traitement du cancer.Accumulating data suggest that both cancer development and recurrence depend on the ability of resistant tumor cells to adopt a quiescent or dormant phenotype following treatment. These dormant cells reside in various tissues of patients in complete remission without any clinical manifestation until they reactivate and cause tumor recurrence. Mechanisms that control the activation of quiescent tumor cells remain poorly understood, however, the tumor microenvironment, cellular interactions and various diffusible factors appear essential. Herein, our goal is to decipher whether a major pro-inflammatory cytokine, Tumor Necrosis Factor (TNF) contributes to the quiescence/activation phenotypic switch in melanoma. For this purpose, we used a 3D melanosphere and the in vivo-like skin equivalent models in which to reconstitute the in vivo-relevant cellular heterogeneity and tumor organization and an inducible histone 2B coupled to the GFP (H2B-GFP) expression system to identify the quiescent cell compartment and to monitor the TNF-induced changes. Our results suggest that TNF increases the proportion of H2B-GFP-positive, label retaining cells (LRC) in melanospheres. The LRCs were enriched in melanoma stem cell markers, ABCB5 and VEGFR and this was upregulated by TNF. Furthermore, TNF increases the number of melanospheres, and in skin equivalents, the presence of TNF seems to inhibit the differentiation of melanoma cells and increase the stem cell compartment. This effect appears to be governed by the activation of the PI3K / Akt pathway. In conclusion, these data show that inflammatory environment induced by TNF, activates melanoma quiescent stem cells and increases the compartment of stem cells in skin equivalents by preventing their differentiation. Therefore, the control of inflammation and signaling pathways involved in the maintenance of tumor dormancy during the treatment of the original tumor would be a good therapeutic strategy in the fight against cancer recurrences.A lot of data suggest that a cancer cell subpopulation is able to enter quiescence in response to cancer therapy, therefore we have also studied the effects of the first targeted therapy of melanoma: vemurafenib, on the stem cell compartment. Our results show that vemurafenib increases the number of melanospheres and the percentage of ABCB5+ cells. So vemurafenib increases the melanoma stem cell compartment. Vemurafenib increases also the percentage of H2B-GFP + cells and the percentage of cells in the GO phase of the cell cycle, so induces quiescence of melanoma cells. We also showed that vemurafenib stimulates activation of proteins regulating quiescence of stem cells.We hope that our research will provide new knowledge about the mechanisms that control the activation of quiescent cancer stem cells and provide new perspectives for the treatment of cancer

La détection des évènements rares

International audienc

La détection des évènements rares

International audienc

Adipose Tissue Properties in Tumor-Bearing Breasts

International audienceThe tissue stroma plays a major role in tumors' natural history. Most programs for tumor progression are not activated as cell-autonomous processes but under the conditions of cross-talks between tumor and stroma. Adipose tissue is a major component of breast stroma. This study compares adipose tissues in tumor-bearing breasts to those in tumor-free breasts with the intention of defining a signature that could translate into markers of cancer risk. In tumor-bearing breasts, we sampled adipose tissues adjacent to, or distant from the tumor. Parameters studied included: adipocytes size and density, immune cell infiltration, vascularization, secretome and gene expression. Adipose tissues from tumor-bearing breasts, whether adjacent to or distant from the tumor, do not differ from each other by any of these parameters. By contrast, adipose tissues from tumor-bearing breasts have the capacity to secrete twice as much interleukin 8 (IL-8) than those from tumor-free breasts and differentially express a set of 137 genes of which a significant fraction belongs to inflammation, integrin and wnt signaling pathways. These observations show that adipose tissues from tumor-bearing breasts have a distinct physiological status from those from tumor-free breasts. We propose that this constitutive status contributes as a non-cell autonomous process to determine permissiveness for tumor growth

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance, and immune evasion. By single-cell RNA-sequencing analysis in PDAC patients, we reveal hereby Solute Carrier Family 4 Member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune-checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an anti-tumor immune response in PDAC

A Novel 8-Predictors Signature to Predict Complicated Disease Course in Pediatric-onset Crohn’s Disease: A Population-based Study

International audienceBackground The identification of patients at high risk of a disabling disease course would be invaluable in guiding initial therapy in Crohn’s disease (CD). Our objective was to evaluate a combination of clinical, serological, and genetic factors to predict complicated disease course in pediatric-onset CD. Methods Data for pediatric-onset CD patients, diagnosed before 17 years of age between 1988 and 2004 and followed more than 5 years, were extracted from the population-based EPIMAD registry. The main outcome was defined by the occurrence of complicated behavior (stricturing or penetrating) and/or intestinal resection within the 5 years following diagnosis. Lasso logistic regression models were used to build a predictive model based on clinical data at diagnosis, serological data (ASCA, pANCA, anti-OmpC, anti-Cbir1, anti-Fla2, anti-Flax), and 369 candidate single nucleotide polymorphisms. Results In total, 156 children with an inflammatory (B1) disease at diagnosis were included. Among them, 35% (n = 54) progressed to a complicated behavior or an intestinal resection within the 5 years following diagnosis. The best predictive model (PREDICT-EPIMAD) included the location at diagnosis, pANCA, and 6 single nucleotide polymorphisms. This model showed good discrimination and good calibration, with an area under the curve of 0.80 after correction for optimism bias (sensitivity, 79%, specificity, 74%, positive predictive value, 61%, negative predictive value, 87%). Decision curve analysis confirmed the clinical utility of the model. Conclusions A combination of clinical, serotypic, and genotypic variables can predict disease progression in this population-based pediatric-onset CD cohort. Independent validation is needed before it can be used in clinical practice