Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. Significance: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.

Background/Objectives: A comparative study of silver (Ag), titanium nitride (TiN), zirconium nitride (ZrN), and copper (Cu) coatings on titanium (Ti) disks, considering the specifications of a microporous skin- and bone-integrated titanium pylon (SBIP), was performed to assess their biocompatibility, osseointegration, and mechanical properties. Methods: To assess cytotoxicity and biocompatibility, Ti disks with various metal coatings were co-cultured with FetMSCs and MG-63 cells for 1, 3, 7, and 14 days and subsequently evaluated using a cell viability assay, as supported by SEM and confocal microscopy studies. The antimicrobial activity of the selected four materials coating the implants was tested against S. aureus by mounting Ti disks onto the surface of LB agar dishes spread with a bacterial suspension and measuring the diameter of the growth inhibition zones. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) analysis of the relative gene expression of biomarkers that are associated with extracellular matrix components (fibronectin, vitronectin, type I collagen) and cell adhesion (α2, α5, αV integrins), as well as of osteogenic markers (osteopontin, osteonectin, TGF-β1, SMAD), was performed during the 14-day follow-up period. Additionally, the activity of matrix metalloproteinases (MMP-1, -2, -8, -9) was assessed. Results: All samples with metal coatings, except the copper coating, demonstrated a good cytotoxicity profile, as evidenced by the presence of a cellular monolayer on the sample surface on the 14th day of the follow-up period (as shown by SEM and inverted confocal microscopy). All metal coatings enhanced MMP activity, as well as cellular adhesion and osteogenic marker expression; however, TiN showed the highest values of these parameters. Significant inhibition of bacterial growth was observed only in the Ag-coated Ti disks, and it persisted for over 35 days. Conclusions: The silver-based coating, due to its high antibacterial activity, low cytotoxicity, and biointegrative capacity, can be recommended as the coating of choice for microporous titanium implants for further preclinical studies.

Oct4 is well established as a core regulator of pluripotency, yet emerging evidence points to an additional role in lineage specification during the exit from the pluripotent state. Although Oct4 expression has been observed in early mesodermal progenitors, its precise function in this developmental context remains unclear. To investigate this, we employed embryoid bodies (EBs) as a model of spontaneous differentiation that recapitulates key aspects of early embryonic development in vitro. In accordance with previous studies, reporter assay revealed a distinct temporal pattern characterized by the strong, transient co-expression of Oct4 and the early mesoderm-specifying marker gene Brachyury within a narrow developmental window, consistent with the Oct4 role in early mesodermal progenitors. We further examined the consequences of the Oct4 loss at early stages of this differentiation. Conditional knockout of the Oct4 gene resulted in a significant reduction in EB size and accumulation of cells in the G0/G1 phase, indicating a critical requirement for Oct4 in maintaining cell proliferation. Despite this defect, cells retained the ability to initiate multilineage differentiation, albeit with reduced expression of Brachyury and elevated expression of endodermal markers FoxA2 and Sox17. Interestingly, the formation of beating cardiomyocyte-like structures was also diminished following Oct4 loss and could not be rescued by simply increasing cell numbers. Taken together, these findings highlight an important Oct4 function in mesodermal differentiation, mediated through the maintenance of proliferative capacity of early mesodermal progenitors.

Motility analysis of sorted C6, T98G, and U251 cell lines by the level of mHsp70 expression. A, Representative scheme of mHsp70 expression analysis and FACS for subsequent determination of cell migration characteristics. B, Confocal microscopy images of C6, T98G, and U251. DAPI was applied for nucleus staining (blue). FITC-IgG1 and FITC-anti-Hsp70 antibody was used for detecting mHsp70 on the plasma membrane (green). Scale bars, 50 μm. C, Mean fluorescence intensity of FITC-anti-Hsp70 antibody in arbitrary units measured by flow cytometry (n = 4). D, Example of tracks rose plots in motility analysis. E, C6, T98G, and U251 cell tracks violin plots. F, Phase contrast live images of wound healing assay of subpopulations with different mHsp70 expression and plots of wound area vs. healing time. Scale bars for images, 400 μm.

Grade content and co-localization of biomarkers (Hsp70, Nestin, SOX2) on histological preparations of human GBM.