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Recently, the need to develop a robust three-dimensional (3D) cell culture system that serves as a valuable in vitro tumor model has been emphasized. This system should closely mimic the tumor growth behaviors observed in vivo and replicate the key elements and characteristics of human tumors for the effective discovery and development of anti-tumor therapeutics. Therefore, in this study, we developed an effective 3D in vitro model of human prostate cancer (PC) using a marine collagen-based biomimetic 3D scaffold. The model displayed distinctive molecular profiles and cellular properties compared with those of the 2D PC cell culture. This was evidenced by (1) increased cell proliferation, migration, invasion, colony formation, and chemoresistance; (2) upregulated expression of crucial multidrug-resistance- and cancer-stemness-related genes; (3) heightened expression of key molecules associated with malignant progressions, such as epithelial–mesenchymal transition transcription factors, Notch, matrix metalloproteinases, and pluripotency biomarkers; (4) robust enrichment of prostate cancer stem cells (CSCs); and (5) enhanced expression of integrins. These results suggest that our 3D in vitro PC model has the potential to serve as a research platform for studying PC and prostate CSC biology, as well as for screening novel therapies targeting PC and prostate CSCs.

Prolonged thymic involution results in decreased thymopoiesis and thymic output, leading to peripheral T-cell deficiency. Since the thymic-dependent pathway is the only means of generating fully mature T cells, the identification of strategies to enhance thymic regeneration is crucial in developing therapeutic interventions to revert immune suppression in immunocompromised patients. The present study clearly shows that fish collagen peptides (FCPs) stimulate activities of thymic epithelial cells (TECs), including cell proliferation, thymocyte adhesion, and the gene expression of thymopoietic factors such as FGF-7, IGF-1, BMP-4, VEGF-A, IL-7, IL-21, RANKL, LTβ, IL-22R, RANK, LTβR, SDF-1, CCL21, CCL25, CXCL5, Dll1, Dll4, Wnt4, CD40, CD80, CD86, ICAM-1, VCAM-1, FoxN1, leptin, cathepsin L, CK5, and CK8 through the NF-κB signal transduction pathway. Furthermore, our study also revealed the cytoprotective effects of FCPs on TECs against cyclophosphamide-induced cellular injury through the NF-κB signaling pathway. Importantly, FCPs exhibited a significant capability to facilitate thymic regeneration in mice after cyclophosphamide-induced damage via the NF-κB pathway. Taken together, this study sheds light on the role of FCPs in TEC function, thymopoiesis, and thymic regeneration, providing greater insight into the development of novel therapeutic strategies for effective thymus repopulation for numerous clinical conditions in which immune reconstitution is required.

Objective: To improve the understanding of the progression and treatment of cancer for the development of more accurate and physiologically relevant in vitro tumor models that serve as important tools in cancer research. Methods: Hydrogels for 3D cell culture were prepared as previously described by our group. To measure the cell proliferation and chemotherapeutic sensitivity, the WST-1 colorimetric assay was performed and colony-forming assay was performed to compare colony growth. Wound-healing assay was performed by making scratch wounds when cells reached confluency and scratch closure was monitored. Hydrogel invasion assay was performed and the depth of cell invasion was observed by cell fluorescence using a confocal laser scanning microscope. Western blot was performed for the detection of apoptotic proteins and key EMT and stemness markers were observed by RT-PCR. Flow cytometry was performed for the detection of cancer stem cell (CSC) population and CSC isolation. Results: 3D ovarian cancer (OC) exhibited biochemical and physiological features, including (1) enhanced cell proliferation, migration and invasion, colony formation, and chemoresistance; (2) suppressed apoptosis with altered expression levels of apoptosis-regulating molecules; (3) upregulated expression of crucial multidrug resistance-related genes; (4) accentuated expression of key molecules associated with malignant progression, such as epithelial--mesenchymal transition transcription factors, Notch, and pluripotency biomarkers; and (5) robust enrichment of ovarian CSCs. Conclusion: An effective 3D in vitro culture method was developed and optimized for growing multicellular OC spheres derived from different OC cell lines. And we demonstrated its usefulness in the isolation and enrichment of ovarian CSCs. [ABSTRACT FROM AUTHOR]

Objective: To improve the understanding of endometriosis progression and treatment for the development of accurate and physiologically relevant in vitro 3D cell culture models, which could serve as important tools in endometriosis research. Methods: A CCK-8 cell proliferation assay and nuclear antigen Ki-67 immunofluorescence staining was performed to assess cell proliferation. The levels of phosphorylated Smad-2 (pSmad-2), transforming growth factor-β (TGF-β) receptor 2 and key epithelial- mesenchymal transition (EMT) and stemness protein expressions were assessed using a western blot assay. For the detection of the expression levels of key genes associated with inflammatory and immunological responses, qRT-PCR was performed. Results: Di-(2-ethylhexyl) phthalate (DEHP) treatment enhanced proliferation, migration, and inflammatory responses, along with EMT and stemness induction in human endometrial and endometriotic cells (EEECs). The selective TGF-βR1/2 inhibitor LY2109761 reversed the DEHP-induced cell proliferation and migration enhancement as well as the increased expression of crucial molecules involved in inflammation, EMT, and stemness, indicating that DEHP-triggered phenomena occur via the TGF-β/Smad signaling pathway. Conclusion: DEHP plays an important role in the etiology and pathogenesis of endometriosis by enhancing cell proliferation and migration and inducing EMT and stem cell properties via the TGF-β/Smad signaling pathway EEECs using a biomimetic hydrogel-based 3D culture model. Furthermore, our findings suggest that the TGF-β/Smad signaling pathway is a potential therapeutic target for DEHP-induced endometriosis. [ABSTRACT FROM AUTHOR]