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 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.

Glutamine deprivation counteracts hypoxia-induced chemoresistance.

The microenvironment of solid tumors is a key determinant of therapy efficacy. The co-occurrence of oxygen and nutrient deprivation is a common phenomenon of the tumor microenvironment and associated with treatment resistance. Cholangiocarcinoma (CCA) is characterized by a very poor prognosis and pronounced chemoresistance. A better understanding of the underlying molecular mechanisms is urgently needed to improve therapy strategies against CCA. We sought to investigate the importance of the conditionally essential amino acid glutamine, a centrally important nutrient for a variety of solid tumors, for CCA. Glutamine levels were strongly decreased in CCA samples and the growth of established human CCA cell lines was highly dependent on glutamine. Using gradual reduction of external glutamine, we generated derivatives of CCA cell lines which were able to grow without external glutamine (termed glutamine-depleted (GD)). To analyze the effects of coincident oxygen and glutamine deprivation, GD cells were treated with cisplatin or gemcitabine under normoxia and hypoxia. Strikingly, the well-established phenomenon of hypoxia-induced chemoresistance was completely reversed in GD cells. In order to better understand the underlying mechanisms, we focused on the oncogene c-Myc. The combination of cisplatin and hypoxia led to sustained c-Myc protein expression in wildtype cells. In contrast, c-Myc expression was reduced in response to the combinatorial treatment in GD cells, suggesting a functional importance of c-Myc in the process of hypoxia-induced chemoresistance. In summary, these findings indicate that the mechanisms driving adaption to tumor microenvironmental changes and their relevance for the response to therapy are more complex than expected.

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family.

Neonates are extremely susceptible to bacterial infections, and evidences suggest that phagocytosis-induced cell death (PICD) is less frequently triggered in neonatal monocytes than in monocytes from adult donors. An insufficient termination of the inflammatory response, leading to a prolonged survival of neonatal monocytes with ongoing proinflammatory cytokine release, could be associated with the progression of various inflammatory diseases in neonates. Our previous data indicate that amphiregulin (AREG) is increasingly expressed on the cell surface of neonatal monocytes, resulting in remarkably higher soluble AREG levels after proteolytic shedding. In this study, we found that E. coli-infected neonatal monocytes show an increased phosphorylation of ERK, increased expression of Bcl-2 and Bcl-XL, and reduced levels of cleaved caspase-3 and caspase-9 compared to adult monocytes. In both cell types, additional stimulation with soluble AREG further increased ERK activation and expression of Bcl-2 and Bcl-XL and reduced levels of cleaved caspase-3 and caspase-9 in an EGFR-dependent manner. These data suggest that reduced PICD of neonatal monocytes could be due to reduced intrinsic apoptosis and that AREG can promote protection against PICD. This reduction of the intrinsic apoptosis pathway in neonatal monocytes could be relevant for severely prolonged inflammatory responses of neonates.

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.

Annexin A1 sustains tumor metabolism and cellular proliferation upon stable loss of HIF1A.

Despite the approval of numerous molecular targeted drugs, long-term antiproliferative efficacy is rarely achieved and therapy resistance remains a central obstacle of cancer care. Combined inhibition of multiple cancer-driving pathways promises to improve antiproliferative efficacy. HIF-1 is a driver of gastric cancer and considered to be an attractive target for therapy. We noted that gastric cancer cells are able to functionally compensate the stable loss of HIF-1α. Via transcriptomics we identified a group of upregulated genes in HIF-1α-deficient cells and hypothesized that these genes confer survival upon HIF-1α loss. Strikingly, simultaneous knock-down of HIF-1α and Annexin A1 (ANXA1), one of the identified genes, resulted in complete cessation of proliferation. Using stable isotope-resolved metabolomics, oxidative and reductive glutamine metabolism was found to be significantly impaired in HIF-1α/ANXA1-deficient cells, potentially explaining the proliferation defect. In summary, we present a conceptually novel application of stable gene inactivation enabling in-depth deconstruction of resistance mechanisms. In theory, this experimental approach is applicable to any cancer-driving gene or pathway and promises to identify various new targets for combination therapies.

Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion.

BACKGROUND: The conventional expansion of human mesenchymal stromal cells (hMSC) for tissue engineering or (pre-) clinical investigation includes the use of 10% fetal bovine serum (FBS). However, there exists immense lot-to-lot variability in FBS samples and time consuming as well as cost intensive lot pre-testing is essential to guarantee optimal hMSC proliferation and stem cells characteristics maintenance. Furthermore, lot-to-lot variability impedes the long-term consistency of research and comparability between research groups. Therefore, we investigated the use of defined, invariable, non-synthetic FBS in low serum culture conditions for isolation and expansion of hMSC. METHODS: hMSC were isolated from bone marrow in Panserin 401 supplemented with growth factors and 2% MSC-tested or non-tested, defined, invariable, non-synthetic FBS and further cultivated in vitro. The surface marker expression, differentiation capacity as well as cell proliferation and cytotoxicity was analyzed and compared between serum samples. RESULTS: Cells isolated and cultivated with low concentrations of MSC-tested or non-tested FBS demonstrated no differences in surface marker expression or differentiation capacity. Proliferation of hMSC was equal in medium supplemented with either serum with no indication of cell death. CONCLUSIONS: The low serum concentration in Panserin 401 supplemented with growth factors enables the use of defined, invariable, non-synthetic FBS for the isolation and expansion of hMSC. The required hMSC characteristics like surface marker expression and differentiation capacity are maintained. Importantly, no differences in the cell proliferation could be detected. Therefore, using these low-serum culture conditions, the need for lot-to-lot pre-testing of FBS usually needed for optimal hMSC expansion is abolished leading to long-term consistency and comparability of results.