Multicellular Modelling of Difficult-to-Treat Gastrointestinal Cancers: Current Possibilities and Challenges

Cancers affecting the gastrointestinal system are highly prevalent and their incidence is still increasing. Among them, gastric and pancreatic cancers have a dismal prognosis (survival of 5-20%) and are defined as difficult-to-treat cancers. This reflects the urge for novel therapeutic targets and aims for personalised therapies. As a prerequisite for identifying targets and test therapeutic interventions, the development of well-established, translational and reliable preclinical research models is instrumental. This review discusses the development, advantages and limitations of both patient-derived organoids (PDO) and patient-derived xenografts (PDX) for gastric and pancreatic ductal adenocarcinoma (PDAC). First and next generation multicellular PDO/PDX models are believed to faithfully generate a patient-specific avatar in a preclinical setting, opening novel therapeutic directions for these difficult-to-treat cancers. Excitingly, future opportunities such as PDO co-cultures with immune or stromal cells, organoid-on-a-chip models and humanised PDXs are the basis of a completely new area, offering close-to-human models. These tools can be exploited to understand cancer heterogeneity, which is indispensable to pave the way towards more tumour-specific therapies and, with that, better survival for patients.Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Targeting endoglin expressing cells in the tumor microenvironment does not inhibit tumor growth in a pancreatic cancer mouse model

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenviron-ment. It is also characterized by high accumulation of dense stroma, composed of mostly cancer-associated fibroblasts (CAFs). Multiple subsets of CAFs are described, with one of them expressing the transforming growth factor (TGF)-beta co-receptor endoglin. In previous work, we and others have shown that endoglin-expressing CAFs stimulate tumor progression and metastasis. Therefore, in this study, we set out to investigate the role of endoglin-expressing CAFs in pancreatic cancer progression. Methods: First, we investigated the expression of endoglin on CAFs in both human tissues as well as a mouse model for PDAC. Since CAF-specific endoglin expression was high, we targeted endoglin by using the endoglin neutralizing antibody TRC105 in the murine KPC model for PDAC. Results: Although some signs of immune activation were observed, TRC105 did not affect tumor growth. Since 90% of the CD8+ T-cells expressed the immune checkpoint PD-1, we investigated the combination with a PD1 checkpoint inhibitor, which did not enhance therapeutic responses. Finally, genetic deletion of endoglin from collagen 1a1 expressing cells also did not affect the growth of the mouse KPC tumors. Conclusion: Our results show that although endoglin is highly expressed on PDAC-CAFs and signaling is efficiently inhibited by TRC105, this does not result in decreased tumor growth in the KPC model for pancreatic cancer.Experimentele farmacotherapi

Dynamic visualization of TGF-beta/SMAD3 transcriptional responses in single living cells

Transforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-beta/SMAD3 signaling kinetics, we developed a dynamic TGF-beta/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-beta/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-beta challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-beta/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes

Dynamic Visualization of TGF-beta/SMAD3 Transcriptional Responses in Single Living Cells

Transforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-beta/SMAD3 signaling kinetics, we developed a dynamic TGF-beta/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-beta/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-beta challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-beta/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.Circadian clocks in health and diseas