Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

Use of non-invasive cardiac monitoring to guide discontinuation of postpartum magnesium sulfate in individuals with preeclampsia with severe features.

OBJECTIVES: To determine the utility of using total peripheral systemic vascular resistance assessed using non-invasive cardiac monitor for individualizing the duration of postpartum magnesium sulfate in individuals with preeclampsia with severe features. STUDY DESIGN: Single center pilot randomized controlled trial in which singleton pregnant individuals with preeclampsia with severe features were randomized to 24 h of postpartum magnesium sulfate per standard of care (control group) or individualized duration of postpartum magnesium sulfate based on reduction in post-delivery systemic vascular resistance (intervention group). Systemic vascular resistance was assessed with non-invasive cardiac monitoring using the Cheetah® system. A 30 % reduction (maintained for 1 h) from baseline post-delivery systemic vascular resistance was used as a cutoff for discontinuation of postpartum magnesium sulfate. Our primary outcome was duration of postpartum magnesium sulfate use in hours. Secondary outcomes included a composite of maternal morbidities associated with preeclampsia. RESULTS: Of 53 individuals enrolled, we excluded 6 from this analysis due to insufficient data to assess primary outcome. Baseline characteristics of the control (n = 26) and intervention (n = 21) groups were similar. Six (28.6 %) individuals in intervention group met the systemic vascular resistance criteria and had their postpartum magnesium sulfate discontinued before 24 h. The duration of postpartum magnesium sulfate infusion was shorter in the intervention group (21.6 ± 4.7 h; range: 7-24 h) compared with control group (24 h, p = 0.02). There was no difference in secondary outcomes between the two groups. There was no difference in adverse outcomes in individuals that had magnesium discontinued earlier than 24 h. CONCLUSION: Non-invasive monitoring of systemic vascular resistance can be a valuable tool to individualize the duration of postpartum magnesium sulfate for preeclampsia with severe features. These findings should be conformed in a larger trial.

Nutrient restriction-activated Fra-2 promotes tumor progression via IGF1R in miR-15a downmodulated pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, characterized by an intense desmoplastic reaction that compresses blood vessels and limits nutrient supplies. PDAC aggressiveness largely relies on its extraordinary capability to thrive and progress in a challenging tumor microenvironment. Dysregulation of the onco-suppressor miR-15a has been extensively documented in PDAC. Here, we identified the transcription factor Fos-related antigen-2 (Fra-2) as a miR-15a target mediating the adaptive mechanism of PDAC to nutrient deprivation. We report that the IGF1 signaling pathway was enhanced in nutrient deprived PDAC cells and that Fra-2 and IGF1R were significantly overexpressed in miR-15a downmodulated PDAC patients. Mechanistically, we discovered that miR-15a repressed IGF1R expression via Fra-2 targeting. In miR-15a-low context, IGF1R hyperactivated mTOR, modulated the autophagic flux and sustained PDAC growth in nutrient deprivation. In a genetic mouse model, Mir15aKO PDAC showed Fra-2 and Igf1r upregulation and mTOR activation in response to diet restriction. Consistently, nutrient restriction improved the efficacy of IGF1R inhibition in a Fra-2 dependent manner. Overall, our results point to a crucial role of Fra-2 in the cellular stress response due to nutrient restriction typical of pancreatic cancer and support IGF1R as a promising and vulnerable target in miR-15a downmodulated PDAC.

Role of Fra-2 in cancer.

Fos-related antigen-2 (Fra-2) is the most recently discovered member of the Fos family and, by dimerizing with Jun proteins, forms the activator protein 1 (AP-1) transcription factor. By inducing or repressing the transcription of several target genes, Fra-2 is critically involved in the modulation of cell response to a variety of extracellular stimuli, stressors and intracellular changes. In physiological conditions, Fra-2 has been found to be ubiquitously expressed in human cells, regulating differentiation and homeostasis of bone, muscle, nervous, lymphoid and other tissues. While other AP-1 members, like Jun and Fos, are well characterized, studies of Fra-2 functions in cancer are still at an early stage. Due to the lack of a trans-activating domain, which is present in other Fos proteins, it has been suggested that Fra-2 might inhibit cell transformation, eventually exerting an anti-tumor effect. In human malignancies, however, Fra-2 activity is enhanced (or induced) by dysregulation of microRNAs, oncogenes and extracellular signaling, suggesting a multifaceted role. Therefore, Fra-2 can promote or prevent transformation, proliferation, migration, epithelial-mesenchymal transition, drug resistance and metastasis formation in a tumor- and context-dependent manner. Intriguingly, recent data reports that Fra-2 is also expressed in cancer associated cells, contributing to the intricate crosstalk between neoplastic and non-neoplastic cells, that leads to the evolution and remodeling of the tumor microenvironment. In this review we summarize three decades of research on Fra-2, focusing on its oncogenic and anti-oncogenic effects in tumor progression and dissemination.