Abscission is the final step of cytokinesis whereby the intercellular bridge (ICB) linking the two daughter cells is cut. The ICB contains a structure called the midbody, required for the recruitment and organization of the abscission machinery. Final midbody severing is mediated by formation of secondary midbody ingression sites, where the ESCRT III component CHMP4B is recruited to mediate membrane fusion. It is presently unknown how cytoskeletal elements cooperate with CHMP4B to mediate abscission. Here, we show that F-actin is associated with midbody secondary sites and is necessary for abscission. F-actin localization at secondary sites depends on the activity of RhoA and on the abscission regulator citron kinase (CITK). CITK depletion accelerates loss of F-actin proteins at the midbody and subsequent cytokinesis defects are reversed by restoring actin polymerization. Conversely, midbody hyperstabilization produced by overexpression of CITK and ANLN is reversed by actin depolymerization. CITK is required for localization of F-actin and ANLN at the abscission sites, as well as for CHMP4B recruitment. These results indicate that control of actin dynamics downstream of CITK prepares the abscission site for the final cut.

Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE’s inhibition could be a therapeutic strategy for MB treatment.

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal components, cancer-associated fibroblasts (CAFs) -- often the most abundant cell type in the tumor microenvironment (TME) -- play a pivotal role in promoting cancer progression through their interactions with tumor cells and other stromal components. Elucidating the mechanisms behind these interactions requires robust methods for isolating and characterizing CAFs, which can also lay the foundation for developing novel CAF-targeted cancer therapies. This study presents a method for isolating fibroblasts from both the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer (PCa) patients. This approach enables the concurrent isolation of CAFs and their normal counterparts (NFs) from the same individual, providing a paired experimental system. Detailed protocols are provided for the maintenance of primary CAFs and NFs for downstream in vitro analyses, as well as for their immortalization to facilitate long-term studies. We also describe functional assays designed to compare the effects of CAFs and NFs on cancer cell behaviors, including proliferation, migration, and anchorage-independent growth. Two complementary approaches are detailed: treatment of cancer cells with fibroblast-derived conditioned media (CM), and direct co-culture of fibroblasts with tumor cells. Together, these methodologies represent a comprehensive toolkit for investigating the dynamic interplay between CAFs and tumor cells, not only in PCa but potentially across a range of human cancers. Moreover, molecular characterization of these interactions may reveal key mediators of CAF-driven oncogenesis, offering promising targets for therapeutic intervention.

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal components, cancer-associated fibroblasts (CAFs) -- often the most abundant cell type in the tumor microenvironment (TME) -- play a pivotal role in promoting cancer progression through their interactions with tumor cells and other stromal components. Elucidating the mechanisms behind these interactions requires robust methods for isolating and characterizing CAFs, which can also lay the foundation for developing novel CAF-targeted cancer therapies. This study presents a method for isolating fibroblasts from both the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer (PCa) patients. This approach enables the concurrent isolation of CAFs and their normal counterparts (NFs) from the same individual, providing a paired experimental system. Detailed protocols are provided for the maintenance of primary CAFs and NFs for downstream in vitro analyses, as well as for their immortalization to facilitate long-term studies. We also describe functional assays designed to compare the effects of CAFs and NFs on cancer cell behaviors, including proliferation, migration, and anchorage-independent growth. Two complementary approaches are detailed: treatment of cancer cells with fibroblast-derived conditioned media (CM), and direct co-culture of fibroblasts with tumor cells. Together, these methodologies represent a comprehensive toolkit for investigating the dynamic interplay between CAFs and tumor cells, not only in PCa but potentially across a range of human cancers. Moreover, molecular characterization of these interactions may reveal key mediators of CAF-driven oncogenesis, offering promising targets for therapeutic intervention.

This study investigates the bactericidal efficacy and penetration ability inside dentinal tubules of calcium hydroxide (CH) and a modified tri-antibiotic paste (ciprofloxacin, metronidazole, clarithromycin) (TAP) during regenerative endodontic procedures (REPs). The blood clot serving as a biological scaffold was introduced into the root canal to assess its influence on bacterial regrowth. Forty-four human extracted teeth were infected with E. faecalis and divided in four experimental groups (N = 10) with positive and negative controls (N = 4). Samples were treated with either CH or TAP as intracanal dressing. Bacterial viability and depth of penetration were evaluated using confocal laser scanning microscopy (CLSM) after fluorescent vital staining. The same analysis was performed with or without blood clot exposure and the data were analyzed by one-way ANOVA and a post hoc Bonferroni test (p < 0.05). TAP demonstrated significantly stronger bactericidal activity than CH (p = 0.008). However, its efficacy significantly decreased in the presence of blood clot (p = 0.032). CH showed a moderate antibacterial effect, with its efficacy reduced in the presence of blood. Blood clot exposure consistently reduced the bactericidal efficacy in both groups (p = 0.01) and the dye penetration in CH group (p = 0.041). In conclusion, TAP demonstrated superior antibacterial performance compared to CH and blood clot exposure seemed to decrease antimicrobial efficacy and depth of disinfection during REPs.