ObjectiveGlioma invasion into surrounding brain tissue drives disease progression but is challenging to evaluate with conventional MRI. This study aimed to determine whether radiomic features extracted from the peritumoral zone capture biologically relevant invasion-associated imaging phenotypes, using membrane-bound Hsp70 (mHsp70) as a molecular correlate.MethodsA retrospective study was performed using preoperative MRI data from 80 patients with glioma, including adult glioblastoma (GBM), adult lower-grade glioma (LGG), and pediatric GBM/ATRT cases. Voxel-wise radiomic features were extracted from the peritumoral region and used to train a stochastic gradient descent (SGD) classifier to distinguish GBM-associated invasion-enriched imaging phenotypes from LGG-associated comparison phenotypes. Patient-level cohort separation was used throughout model development to prevent data leakage. Feature importance was assessed using SHAP analysis. In a biologically validated subgroup of 23 patients, membrane-bound Hsp70 (mHSP70) expression was quantified in peritumoral tissue samples using confocal microscopy. Patient-level radiomic invasion metrics were correlated with mHsp70 fluorescence intensity.ResultsThe SVM classifier demonstrated good discriminative performance, achieving an AUC of 0.875 (95% CI: 0.736–0.896) in the independent test cohort. SHAP analysis identified higher-order texture features, including GLCM-, GLRLM-, and LoG-derived radiomic descriptors, as major contributors to model predictions. Reproducibility analysis demonstrated good-to-excellent agreement for all 20 SHAP-selected features, with mean ICC values ranging from 0.859 to 0.986. In the biological validation subgroup, the radiomics-derived Invasion Burden Index (IBI) showed a significant positive correlation with mHsp70 expression (Spearman ρ = 0.67, p = 0.0004, 95% CI: 0.232–0.805, n = 23). Leave-one-out sensitivity analysis demonstrated stable correlations, indicating that the observed association was not driven by individual cases. Regions exhibiting elevated invasion-associated radiomic signatures frequently extended beyond the conventional MRI-defined tumor margin and showed spatial correspondence with areas of subsequent tumor progression on follow-up imaging.ConclusionsVoxel-wise radiomic analysis of the peritumoral zone identifies invasion-associated imaging phenotypes that correlate with membrane Hsp70 expression, a biological marker associated with aggressive tumor behavior. These findings support the potential utility of radiomic invasion mapping as a non-invasive tool for characterizing infiltrative glioma biology and generating hypotheses regarding patterns of tumor progression. Further prospective studies with spatially matched biological validation are needed.

ObjectiveRecurrent intracranial meningiomas are a significant therapeutic challenge due to their invasive growth and high recurrence risk after surgery and radiotherapy. This study investigates the feasibility of a novel integrated approach combining 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) with intraoperative photodynamic therapy (PDT) for recurrent atypical and anaplastic meningiomas.MethodsIn a single-center, prospective cohort study, 23 patients with recurrent atypical and anaplastic meningiomas received the experimental treatment protocol (FGS+PDT). A retrospective control group (n=35) underwent conventional microsurgery. The intervention included preoperative 5-ALA administration, FGS with visual (Fluorescence Intensity Score, FIS) and quantitative biospectroscopy (Fluorescence Index, FI) guidance, tumor resection, and subsequent PDT (635 nm laser) applied to the resection cavity and tumor matrix. Biospectroscopy guided PDT endpoint (photobleaching and decreasing of FI). Primary outcomes included feasibility, safety, and extent of resection (Simpson Grade), short follow-up period. Histopathological and immunofluorescence analyses of paired pre-/post-PDT biopsies assessed biological effects.ResultsThe FGS+PDT protocol was successfully completed in all patients with an excellent safety profile; no adverse events were attributed to 5-ALA or PDT. All tumors exhibited visible 5-ALA fluorescence. Gross-total resection (Simpson I-II) was achieved in 95.6% (22/23) of the study group versus 77.1% (27/35) in controls (p<0.05). Biospectroscopy revealed significant PpIX accumulation even in visually low-fluorescence tumors. Over a median follow-up of 16 months, no recurrences were observed in the experimental group. Histopathological analysis demonstrated profound PDT-induced effects, including total ablation of progesterone receptor expression in the tumor matrix and a significant increase in caspase-3-mediated apoptosis in the peritumoral zone (36.3 ± 9.6 vs. 14.8 ± 2.2 cells/mm², p<0.0001). Confocal microscopy confirmed subcellular damage, including mitochondrial dysfunction, nuclear degradation, and Hsp70 overexpression.ConclusionThe integrated FGS and PDT protocol is feasible, safe, and demonstrates compelling preliminary efficacy for recurrent atypical and anaplastic meningiomas. It enhances resection and induces profound cytotoxic and apoptotic effects in the residual tumor bed and peritumoral zone. These results need to be further validated in larger, randomized controlled trials.

Characteristics of adult patients with high-grade gliomas.

Motility of Hsp70-positive cells in a GBM tumor sample detected using laser scanning confocal microscopy during observation for 1 h.

Motility of Hsp70-positive cells emerging from a piece of tumor during cultivation for three days.

Analysis of the mass-spectrometry data from isolated lipid rafts.

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

One of the promising targets for fluorescence-guided surgery of malignant brain tumors is a 70 kDa heat shock protein (mHsp70), which has been found on the plasma membrane of tumor cells. In this study, we conjugated the RAS70 peptide, which targets mHsp70 on tumor cells, with the fluorophore Cy7.5 and used it for epifluorescence detection of mHsp70-positive glial tumors and brain metastases. The study included adult patients with newly diagnosed glioblastoma (GBL, n = 7) and brain metastases (MTS, n = 8). Three hours prior to surgery, patients received an oral solution of 5-aminolevulinic acid (5-ALA, 20 mg/kg). During surgery, tumor samples were obtained from three zones: necrotic, viable tumor, and perifocal. Some samples were treated ex vivo with the RAS70-Cy7.5 peptide, while others were treated with the control peptide NGL-RGD-Cy7.5. Epifluorescence images were obtained using an operating microscope in FL400 and FL800 modes, image analysis was performed in ImageJ with calculation of the target-to-background ratio (TBR). The presence of mHsp70 on the tumor cell membrane was additionally determined using immunofluorescence microscopy. Immunofluorescence staining revealed mHsp70-positive tumor cells in all studied samples, with preferential localization observed in the viable (contrast-enhancing) and perifocal tumor zones. TBRs for fluorescence imaging of RAS70-Cy7.5 peptide were significantly higher than those for the control peptide (p < 0.0001): necrotic zone 19.2 a.u. (15.5–21.0), viable tumor 9.0 a.u. (7.0–11.5), perifocal zone 8.9 a.u. (6.7–11.4), control – 1.2 a.u. (0.9–1.7). RAS70 exhibited 37.5

Motility of Hsp70-positive cells in a tumor sample.

Confocal microscopy images of primary glioblastoma cells from patients stained for mHsp70 and Nestin.

Characteristics of patients with brain metastases.

Multiforme glioblastoma-homing peptides, particularly targeting plasma membrane-bound heat shock protein mHsp70, demonstrate great application potential for tumor theranostics. In the current study, to further increase the bioavailability as well as penetration capacity through the blood-brain barrier (BBB) of the mHsp70-targeted peptide TKDNNLLGRFELSG, which is known to bind to the oligomerization sequence of mHsp70 chaperone, the latter was conjugated with tripeptide RGD (forming chimeric peptide termed RAS70). In the model BBB system RAS70 efficiently crossed the barrier accumulating in the glioblastoma cells. Subsequently, in the orthotopic glioma models, intravenous administration of the fluorescently labeled agent (RAS70-sCy7.5) resulted in the tumor retention of peptide (further confirmed by histological studies). Thus, as shown by the biodistribution studies employing epifluorescence imaging, accumulation of RAS70-sCy7.5 in C6 glioma was significantly enhanced as compared to scramble peptide. Local application of the RAS70-sCy7.5 peptide that was sprayed over the dissected brain tissues helped to efficiently delineate the tumors in glioma-bearing animals employing an intraoperative fluorescent imaging system. Tumor-specific internalization of the peptide was further confirmed on the ex vivo primary GBM samples obtained from adult neurooncological patients. In conclusion, RAS70 peptide demonstrated high glioma-homing properties which could be employed for the intraoperative tumor visualization as well as for developing a potential carrier for drug delivery.

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.

Heat shock proteins (HSP), particularly 70 kDa heat shock protein (Hsp70), apart from their cytoplasmic localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-bound protein mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of adult and pediatric brain tumor samples (n=32) showed mHsp70 overexpression on the membrane, especially in the perifocal zone. Mass-spectrometry analysis of lipid rafts isolated from brain tumor cells confirmed the presence of the protein in HSP cluster (including representatives of other families such as HSP70, HSP90), which in turn, during interactome analysis, was associated with proteins involved in cell migration and invasion (i.e., Rac1, RhoC, myosin-9). Application of small-molecule inhibitors of HSP70 (JG-98, PES) led to a significant 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 chaperone inhibitors in orthotopic brain tumors model in rodents significantly decreased tumor progression, which was accompanied by an increased overall survival. Data demonstrate that chaperone inhibitors, particularly JG-98, 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. Apart from chaperone inhibitors we developed mHsp70-targeted peptide that could efficiently cross the blood-brain barrier accumulating in the glioblastoma cells in vivo. Subsequently, in the orthotopic GL261 mice and C6 rat glioma, intravenous administration of the fluorescently labeled peptide resulted in the tumor retention of agent (further confirmed by histological studies) 24 hours following i.v. injection. Topical application of the mHsp70-targeted peptide (sprayed over the freshly dissected brain tissues) helped to delineate the tumors in glioma-bearing animals employing intraoperative fluorescent imaging system. In conclusion, peptide recognizing mHsp70 demonstrated high targeting properties which could be employed for the intraoperative visualization as well as for developing of a potential carrier for drug delivery. Maxim Shevtsov, Natalia Yudintceva, Danila Bobkov, Ruslana Likhomanova, Anastasiya Nechaeva, Anastasiya Lukacheva, Natalia Mikhailova, Viacheslav Fedorov, Elena Oganesyan, Oleg Rozanov, Konstantin Samochernykh, Alexey Ulitin, Alexander Kim, Elena Mikhailova, Rustam Ziganshin, Emil Pitkin, Stephanie E. Combs. Membrane-bound heat shock protein Hsp70 as a new target in oncology: from theory to practice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 57

Gating of mHsp70High cells (field R2) and mHsp70Low cells (field R3) by FACS.

Effect of Hsp70 inhibitors PES and JG-98 on cell viability analyzed using the MTT assay.

Comparison of mean speed and track straightness on various matrices (PL, MG, FN) of patient-derived (non-sorted into high- and low-speed subpopulations) brain tumor cells treated with 1 µM PES or 50 nM JG-98.

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

Confocal microscopy images of primary glioblastoma cells stained for mHsp70 and SOX2.

Motility of Hsp70-positive cells in a GBM tumor sample detected using laser scanning confocal microscopy during observation for 1 h, zoomed field of view from the center of the Video S1 frame.