Adaption to hypoxia and nutrient depletion determines therapy sensitivity of cholangiocarcinoma

A key challenge in the development of effective therapies for cholangiocarcinoma (CCA) is the natural anatomic diversity and molecular heterogeneity of the tumor. The majority of CCA patients present with unresectable tumors and palliative chemotherapy with gemcitabine and/or cisplatin is the current gold standard at this stage. A better understanding of the underlying resistance mechanisms is mandatory to improve therapy strategies. One factor known to profoundly influence susceptibility to anti-cancer drugs is the tumor microenvironment. A co-occurrence of oxygen and nutrient deprivation is a common phenomenon in core regions of solid tumors and both are known to stimulate aggressiveness and treatment resistance. As the non-essential amino acid glutamine plays a central role for the metabolism of a variety of solid tumors, the aim of this thesis was to investigate its general importance for CCA as well as its effect on cytostatic drug therapy in a setting with coincident hypoxia. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) analysis revealed glutamine to be strongly depleted in CCA patient tissue. Moreover, in vitro experiments showed that CCA cell lines are highly dependent on glutamine supply. By using gradual reduction of external glutamine, two extrahepatic CCA (eCCA) cell lines (EGI-1 and TFK-1) were able to overcome glutamine addiction. The glutamine-deprived cell lines obtained in the selection process (termed EGI-1 GD and TFK-1 GD) were capable of glutamine-independent growth. Bioenergetic analysis showed that glutamine deprivation affected mitochondrial respiration of TFK-1 cells, suggesting a shift to glycolysis during the adaption process. Since the effects of coincident hypoxia and long-term glutamine starvation remain broadly unknown, GD cell lines were treated with cisplatin or gemcitabine under normoxia and hypoxia. Under normoxic conditions, GD cells displayed decreased sensitivity to both drugs. Under hypoxia, both parental cell lines showed enhanced chemoresistance, a phenomenon well in line with published literature. Strikingly, hypoxia was not able to increase therapy resistance in GD cells. These results argue for an interconnection between the molecular mechanisms that govern adaptation to glutamine deprivation and the hypoxic response that was not previously appreciated.The oncoprotein c-Myc plays an important role in glutamine metabolism of cancer cells and glutamine deprivation resulted in a reduction of c-Myc protein levels in eCCA cell lines. Glutamine-deprived GD cells showed normalisation of c-Myc to the level of glutamine-supplemented parental cells. When treated with cisplatin, both parental cell lines showed equal levels of c-Myc independent of oxygen availability, whereas distinct hypoxia-mediated downregulation was observed in both GD cell lines. These findings indicate, that c-Myc could be involved in the different cisplatin-induced DNA damage response between cells adapted to glutamine deprivation and their parental counterparts.Given the challenging anatomic diversity of CCA, another aim was to comparatively analyse the response of eCCA and intrahepatic CCA (iCCA) cell lines to cytostatic drug treatment. Whereas both eCCA cell lines showed significant susceptibility to hypoxic conditions, the tested iCCA cell lines (CC-LP-1 and SNU-1079) showed no difference in proliferation. The experiments also revealed a higher tolerance to cisplatin and gemcitabine treatment in iCCA compared to eCCA cell lines with two to ten times higher IC50 values. Moreover, hypoxia-mediated resistance to cisplatin was not detectable in iCCA cell lines. γH2AX is a DNA damage marker which can help to predict tumor response particularly to cisplatin treatment. Analysis of a CCA patient cohort revealed a significant correlation between cytostatic drug treatment and γH2AX-positive tumors as well as a trend towards an improved survival rate in patients with Klatskin tumors that was not detectable in iCCA patients. In summary, the findings suggest that long-term glutamine deprivation does not intensify the resistance-mediating effect of hypoxia, indicating that the adaption to tumor microenvironmental changes might be more complex for therapy outcomes than expected. Moreover, the results indicate that the reliance on oxygen might be dependent on the anatomic origin of the CCA. Since both subclasses showed different susceptibility to anti-cancer drugs, it is reasonable to expect that personalised treatment options are able to improve outcomes

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

Neonates are highly susceptible to microbial infections which is partially attributable to fundamental phenotypic and functional differences between effector cells of the adult and neonatal immune system. The resolution of the inflammation is essential to return to tissue homeostasis, but given that various neonatal diseases, such as periventricular leukomalacia, necrotizing enterocolitis, or bronchopulmonary dysplasia, are characterized by sustained inflammation, newborns seem predisposed to a dysregulation of the inflammatory response. Targeted apoptosis of effector cells is generally known to control the length and extent of the inflammation, and previous studies have demonstrated that phagocytosis-induced cell death (PICD), a special type of apoptosis in phagocytic immune cells, is less frequently triggered in neonatal monocytes than in adult monocytes. We concluded that a rescue of monocyte PICD could be a potential therapeutic approach to target sustained inflammation in neonates. The EGFR ligand amphiregulin (AREG) is shed in response to bacterial infection and was shown to mediate cellular apoptosis resistance. We hypothesized that AREG might contribute to the reduced PICD of neonatal monocytes by affecting apoptosis signaling. In this study, we have examined a cascade of signaling events involved in extrinsic apoptosis by using a well-established in vitro E. coli infection model in monocytes from human peripheral blood (PBMO) and cord blood (CBMO). We found that CBMO shows remarkably higher pro-AREG surface expression as well as soluble AREG levels in response to infection as compared to PBMO. AREG increases intracellular MMP-2 and MMP-9 levels and induces cleavage of membrane-bound FasL through engagement with the EGF receptor. Our results demonstrate that loss of AREG rescues PICD in CBMO to the level comparable to adult monocytes. These findings identify AREG as a potential target for the prevention of prolonged inflammation in neonates

Un hijo del limo: el lugar de la poesía y la militancia en la obra de Miguel Ángel Bustos

Uno de los aspectos centrales en la academia argentina del nuevo milenio, consecuencia del golpe de Estado cívico militar eclesial de 1976, fue el estallido y la persistencia de estudios tanto disciplinares como interdisciplinares en torno a la relación entre política y poesía. Auge que se articula con la abundancia de la producción científica sobre la memoria y más tardíamente entre ésta, la agenda de DDHH y la literatura. Nombres como los de Juan Gelman, Alejandra Pizarnik, Néstor Perlongher, Francisco Urondo, Roberto Santoro comienzan a ser objetos de estudio de la crítica. En lo tocante a la producción de Miguel Ángel Bustos, contemporáneo a la formación de los poetas de la Generación del 60, no ha sido objeto de estudios académicos, salvo el de Ana Porrúa mientras que he podido ubicar notas como las de Susana Cella, Julián Axat y Daniel Freidemberg, entre otros. Miguel Ángel Bustos fue detenido el 30 de mayo de 1976, y sus restos identificados en el año 2014. Periodista, docente universitario, militante del Partido Revolucionario de los Trabajadores, y poeta prolífico. Su producción tanto poética como periodística ha sido reunida y publicada por su hijo Emiliano Bustos. En esta dirección, me detendré en la constitución del campo cultural, en el interregno 60-70, para luego desplegar mi hipótesis de lectura referida a su obra como una discontinuidad con respecto a la poesía en boga en el interregno 60- 70 del siglo XX. Finalmente, enfatizaré su texto El Himalaya o la moral de los pájaros (1970).One of them aspects central in the academy argentina of the new millennium, result of the coup of State civic military ecclesial of 1976, was the pop and the persistence of studies both disciplinary as interdisciplinary on the relationship between political and poetry. Boom that articulates with the abundance of scientific production on memory and later between this, the human rights and literature. Names like Juan Gelman, Alejandra Pizarnik, Néstor Perlongher, Francisco Urondo, Roberto Santoro began to be objects of study of criticism. With regard to the production of Miguel Ángel Bustos, contemporary to the formation of the poets of the generation of the 60, has not been subject to academics, except for the case of Ana Porrúa while I've been able to locate notes as Susana Cella, Julián Axat and Daniel Freidemberg, among others. Miguel Ángel Bustos was arrested on 30 May 1976, and their remains identified in 2014. Journalist, teacher College, militant of Partido Revolucionario de los Trabajadores, and a prolific poet. Its production both poetic as journalism has been gathered and posted by his son Emiliano Bustos. In this address, I will stop in the constitution of the field cultural, in the interregnum 60-70, for then deploy my hypothesis of reading referred to his work as a discontinuity with respect to the poetry in boga in the interregnum 60-70 of the century XX. Finally, I am your text El Himalaya o la moral de los pájaros (1970)

Patient‐specific logic models of signaling pathways from screenings on cancer biopsies to prioritize personalized combination therapies

Mechanistic modeling of signaling pathways mediating patient-specific response to therapy can help to unveil resistance mecha-nisms and improve therapeutic strategies. Yet, creating suchmodels for patients, in particular for solid malignancies, is chal-lenging. A major hurdle to build these models is the limited mate-rial available that precludes the generation of large-scaleperturbation data. Here, we present an approach that couplesex vivohigh-throughput screenings of cancer biopsies usingmicrofluidics with logic-based modeling to generate patient-specific dynamic models of extrinsic and intrinsic apoptosis signal-ing pathways. We used the resulting models to investigate hetero-geneity in pancreatic cancer patients, showing dissimilaritiesespecially in the PI3K-Akt pathway. Variation in model parametersreflected well the different tumor stages. Finally, we used ourdynamic models to efficaciously predict new personalized combi-natorial treatments. Our results suggest that our combination ofmicrofluidic experiments and mathematical model can be a noveltool toward cancer precision medicine

Patient-specific logic models of signaling pathways from screenings on cancer biopsies to prioritize personalized combination therapies

\u3cp\u3eMechanistic modeling of signaling pathways mediating patient-specific response to therapy can help to unveil resistance mechanisms and improve therapeutic strategies. Yet, creating such models for patients, in particular for solid malignancies, is challenging. A major hurdle to build these models is the limited material available that precludes the generation of large-scale perturbation data. Here, we present an approach that couples ex vivo high-throughput screenings of cancer biopsies using microfluidics with logic-based modeling to generate patient-specific dynamic models of extrinsic and intrinsic apoptosis signaling pathways. We used the resulting models to investigate heterogeneity in pancreatic cancer patients, showing dissimilarities especially in the PI3K-Akt pathway. Variation in model parameters reflected well the different tumor stages. Finally, we used our dynamic models to efficaciously predict new personalized combinatorial treatments. Our results suggest that our combination of microfluidic experiments and mathematical model can be a novel tool toward cancer precision medicine.\u3c/p\u3

Supplementary Archive 2

Files included in Supplementary Archive 2, see Evangelista_et_al_README_DRYAD.pd