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.

Over the past several decades, nanocarriers have demonstrated diagnostic and therapeutic (i.e., theranostic) potencies in translational oncology, and some agents have been further translated into clinical trials. However, the practical application of nanoparticle-based medicine in living organisms is limited by physiological barriers (blood–tissue barriers), which significantly hampers the transport of nanoparticles from the blood into the tumor tissue. This review focuses on several approaches that facilitate the translocation of nanoparticles across blood–tissue barriers (BTBs) to efficiently accumulate in the tumor. To overcome the challenge of BTBs, several methods have been proposed, including the functionalization of particle surfaces with cell-penetrating peptides (e.g., TAT, SynB1, penetratin, R8, RGD, angiopep-2), which increases the passing of particles across tissue barriers. Another promising strategy could be based either on the application of various chemical agents (e.g., efflux pump inhibitors, disruptors of tight junctions, etc.) or physical methods (e.g., magnetic field, electroporation, photoacoustic cavitation, etc.), which have been shown to further increase the permeability of barriers.

The plasma membrane (PM) of eukaryotic cells plays a key role in the response to stress, acting as the first line of defense against environmental changes and protecting cells against intracellular perturbations. In this work, we explore how membrane-bound chaperones and membrane lipid domains work together to shape plasma membrane properties—a partnership we refer to as the “epichaperome–plasma membrane lipid axis.” This axis influences membrane fluidity, curvature, and domain organization, which in turn shapes the spatial and temporal modulation of signaling platforms and pathways essential for maintaining cellular integrity and homeostasis. Changes in PM fluidity can modulate the activity of ion channels, such as transient receptor potential (TRP) channels. These changes also affect processes such as endocytosis and mechanical signal transduction. The PM proteome undergoes rapid changes in response to membrane perturbations. Among these changes, the expression of heat shock proteins (HSPs) and their accumulation at the PM are essential mediators in regulating the physical state and functional properties of the membrane. Because of the pivotal role in stress adaptation, HSPs influence a wide range of cellular processes, which we grouped into three main categories: (i) mechanistic insights, differentiating in vitro (liposome, reconstituted membrane systems) and in vivo evidence for HSP-PM recruitment; (ii) functional outputs, spanning how ion channels are affected, changes in membrane fluidity, transcytosis, and the process of endocytosis and exosome release; and (iii) pathological effects, focusing on how rewired lipid–chaperone crosstalk in cancer drives resistance to drugs through altered membrane composition and signaling. Finally, we highlight Membrane Lipid Therapy (MLT) strategies, such as nanocarriers targeting specific PM compartments or small molecules that inhibit HSP recruitment, as promising approaches to modulate the functional stability of epichaperome assembly and membrane functionality, with profound implications for tumorigenesis.

The sequence of constructing a phenotyping map for histological sections of human GBM.

Evaluation of biomarkers co-localization.

Evaluation of the membrane-bound Hsp70 expression in malignant brain tumors by live-cell confocal microscopy imaging. A, Schematic description of the study of brain tumor samples to determine the expression of membrane-associated chaperone in cancer cells, followed by mass spectrometry analysis of isolated lipid rafts. B, Representative confocal microscopy images of the brain tumor samples stained for Hsp70 (green), TMRM (tetramethylrodamine methyl ester; red), disialoganglioside GD2 (red), and protoporphyrin IX (pPIX; magenta). Nuclei were stained by DAPI. Scale bars, 100 μm. Respective MR images (postcontrast T1-weighted and FLAIR images) of the patients are presented. C, Representative confocal microscopy images of the brain metastasis stained for Hsp70 (green), TMRM (tetramethylrodamine methyl ester; red). Nuclei were stained by DAPI (blue). Scale bars, 100 μm. Respective MR images (postcontrast T1-weighted and FLAIR images) of the patients as well as hematoxylin and eosin staining of tumor samples are presented. D, Top row—Fluorescence intensity measurements of mHsp70 expression in live-cell microscopy images of patients with GBM (n = 11) with subsequent Benjamini–Yekutieli test for multiple pairwise comparison of protein expression levels. The bar graph shows the median ± 95% CI (Man–Whitney test result—****, P < 0.0001). The heat map shows P values as the result of a post hoc multiple comparison test. Bottom row—Fluorescence intensity measurements of mHsp70 expression in live-cell microscopy images of brain metastasis (n = 3) with subsequent Benjamini–Yekutieli test for multiple pairwise comparisons of protein expression levels. The bar graph shows the median ± 95% CI (Man–Whitney test result—****, P < 0.0001). The heat map shows P values as the result of a post hoc multiple comparison test. E, Representative multiplex analysis of the immunofluorescence of brain tumor sample stained for Hsp70, SOX2, and nestin. Nuclei were stained by DAPI. NA, necrotic area; VT, vital tissues. Scale bars, 200 μm. F, Representative confocal microscopy images of the normal brain tissue samples stained for Hsp70 (green) and TMRM (tetramethylrodamine methyl ester; red). Nuclei were stained by DAPI (blue). Scale bars, 100 μm. Respective MR images of the patients are presented. G, Live-confocal microscopy images of mHsp70-positive tumor cells in oncostreams detected in GBM samples. Tumor sections were stained for Hsp70 (green), TMRM (red), and DAPI (blue) for nuclei. Scale bars, 100 μm. For the descriptive characteristics of patients, see Supplementary Table S1.