Development of a 3D Tumor Spheroid Model from the Patient's Glioblastoma Cells and Its Study by Metabolic Fluorescence Lifetime Imaging.

Patient-specific in vitro tumor models are a promising platform for studying the mechanisms of oncogenesis and personalized selection of drugs. In case of glial brain tumors, development and use of such models is particularly relevant as the effectiveness of such tumor treatment remains extremely unsatisfactory. The aim of the study was to develop a model of a 3D tumor glioblastoma spheroid based on a patient's surgical material and to study its metabolic characteristics by means of fluorescence lifetime imaging microscopy of metabolic coenzymes. Materials and Methods: The study was conducted with tumor samples from patients diagnosed with glioblastoma (Grade IV). To create spheroids, primary cultures were isolated from tumor tissue samples; the said cultures were characterized morphologically and immunocytochemically, and then planted into round-bottom ultra low-adhesion plates. The number of cells for planting was chosen empirically. The characteristics of the growth of cell cultures were compared with spheroids from glioblastomas of patients with U373 MG stable line of human glioblastoma. Visualization of autofluorescence of metabolic coenzymes of nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) in spheroids was performed by means of an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany). The autofluorescence decay parameters were studied under normoxic and hypoxic conditions (3.5% О2). Results: An original protocol for 3D glioblastoma spheroids cultivation was developed. Primary glial cultures from surgical material of patients were obtained and characterized. The isolated glioblastoma cells had a spindle-shaped morphology with numerous processes and a pronounced granularity of cytoplasm. All cultures expressed glial fibrillary acidic protein (GFAP). The optimal seeding dose of 2000 cells per well was specified; its application results in formation of spheroids with a dense structure and stable growth during 7 days. The FLIM method helped to establish that spheroid cells from the patient material had a generally similar metabolism to spheroids from the stable line, however, they demonstrated more pronounced metabolic heterogeneity. Cultivation of spheroids under hypoxic conditions revealed a transition to a more glycolytic type of metabolism, which is expressed in an increase in the contribution of the free form of NAD(P)H to fluorescence decay. Conclusion: The developed model of tumor spheroids from patients' glioblastomas in combination with the FLIM can serve as a tool to study characteristics of tumor metabolism and develop predictive tests to evaluate the effectiveness of antitumor therapy.

[A tool to assess functional communication skills and its correspondence with traditional language testing in patients with brain tumors]. / Instrument izmereniya funktsional'nykh kommunikativnykh navykov i sopostavlenie ego s traditsionnym rechevym testirovaniem u patsientov s opukholyami golovnogo mozga.

OBJECTIVE: To identify the relationship between speech impairment as measured by the Russian Aphasia Test (RAT) and functional communication as assessed by the Communicative Effectiveness Index (CETI). MATERIAL AND METHODS: RAT and CETI were administered to 87 patients at two time points, before surgery and in 3 months after brain tumor resection surgery. RESULTS: There were significant correlations between CETI and the total scores on RAT subtests for speech comprehension and production before surgery but not in the follow-up period. CONCLUSION: The present research is the first to present the Russian version of CETI and to confirm the relationship between speech disorders measured by the comprehensive standardized battery for evaluating speech function RAT and functional communication as measured by CETI.

Personalized Medicine in Brain Gliomas: Targeted Therapy, Patient-Derived Tumor Models (Review).

Gliomas are the most common type of primary malignant brain tumors. The choice of treatments for these tumors was quite limited for many years, and therapy results generally remain still unsatisfactory. Recently, a significant breakthrough in the treatment of many forms of cancer occurred when personalized targeted therapies were introduced which inhibit tumor growth by affecting a specific molecular target. Another trend gaining popularity in oncology is the creation of patient-derived tumor models which can be used for drug screening to select the optimal therapy regimen. Molecular and genetic mechanisms of brain gliomas growth are considered, consisting of individual components which could potentially be exposed to targeted drugs. The results of the literature review show a higher efficacy of the personalized approach to the treatment of individual patients compared to the use of standard therapies. However, many unresolved issues remain in the area of predicting the effectiveness of a particular drug therapy regimen. The main hopes in solving this issue are set on the use of patient-derived tumor models, which can be used in one-stage testing of a wide range of antitumor drugs.

Application of 2D Gait Analysis for the Assessment of Gait Disturbance in Patients with Spastic Tetraparesis.

The aim of the study was to explore the use of 2D gait analysis for assessing gait abnormalities in patients with spastic tetraparesis associated with spinal cord injury and other lesions of the cervical spinal cord. Materials and Methods: The study included 12 patients with tetraparesis of various etiologies. Gait assessment was performed by video analysis using reflective markers (1.5 cm) and a special walking platform. The spatial coordinates of the markers were determined by capturing the reflected light with infrared LEDs located around the lenses of video cameras. Results: Using 2D gait analysis, numerical indicators of gait disturbance in spastic tetraparesis were obtained. We found a prolongation of the stand phase with a shortening of the swing phase (from 81.9 [76.1; 89.2] to 85.3 [74.4; 90.2]%; p=0.97) and the period of the double step (from 0.50 [0.45; 0.96] to 0.40 [0.34; 0.66]; p=0.4) in comparison with the target (normal) values (60% - for the stand phase; 1.41 - for the double-step period). The movements in the hip, knee, and ankle joints are described using numerical values.We then compared the data obtained from the left and right sides of the patient's body: there were no statistically significant differences between the two sets of data. We also compared the gait characteristics before and after treatment (in 4 patients). Statistically significant differences in values were obtained for the stand and swing phases (p=0.035), the range of motion in the hip joint (p=0.01), and gait velocity (p=0.046). Kendall's analysis revealed no significant correlation between the data obtained by video gait analysis and the gait changes by the Modified Ashworth Scale (р>0.05). Conclusion: 2D gait analysis is a promising method for quantifying gait disturbance in patients with spastic tetraparesis. It allows one to identify characteristic gait patterns, in particular, an increase in the stand phase with a shortening of the swing phase and the double step period, as well as a decrease in the range of motion in the hip joints with an increase in the knee and ankle ones.

[Cytologic control of the resection margins of hemispheric gliomas and metastases]. / Tsitologicheskii kontrol' granits rezektsii polusharnykh gliom i metastazov.

INTRODUCTION: Postoperative MRI is the conventional method for assessing the radicalness of hemispherical tumors excision, but the method has limitations on sensitivity in the assessment of tumor infiltration of the peritumoral zone. The 'gold standard' for detecting tumor cells is the microscopic visualisation. AIM: To study the possibilities of a cytological study of the excision margins of glial and metastatic tumors for an objective assessment of the radical nature of the operation. MATERIAL AND METHODS: The study included 35 patients with intracerebral tumors who underwent open surgery at a university clinic of Volga Research Medical University in 2018-2019: 15 patients with metastasis, 13 patients with MRI-contrasting gliomas Grade III-IV and 7 patnents with non-MRI-contrasting gliomas Grade II-III. During the surgery, samples for cytological examination were taken from the following sites: from the tumor, from the nearest perifocal zone, and at a distance of 5-7 mm, along the border of the extended resection. 154 samples were examined: from 2 to 5 for each patient. RESULTS: The data on the radicalness of the operation, obtained by methods of cytological analysis of the resection margins and postoperative MRI, are not only consistent (p=0.001), but also complement each other, in particular, in some cases, tumor cells were found even in those areas where the tumor tissue was not detected with MRI. In cases of cerebral metastases excision, tumor cells in the nearest perifocal zone were found in 8 out of 28 samples (28.6%), at the extended resection margins - in 3 out of 29 (10.3%). In cases of resection of MRI-contrasting gliomas Grade III-IV, tumor cells in the nearest perifocal zone were found in 22 out of 32 samples (68.8%), at the extended resection margins - in 14 out of 20 (70%). In cases of excision of diffuse gliomas Grade II-III, tumor cells in the nearest perifocal zone were found in 10 out of 17 samples (58.9%), at the extended resection margins - in 4 out of 11 (36.4%). CONCLUSION: The first data obtained demonstrated sufficient informativeness of the cytologic examination of the peritumoral zone as an additional tool for assessing the radicalness of glioma and metastasis surgery. Cytologic analysis of the perifocal zone shows that the extension of the borders of the removal of Grade III-IV gliomas has no advantages, because tumor cells were found both in the nearest perifocal zone and at the extended resection margins with with approximately the same frequency.

Endogenous and Exogenous Factors Affecting the Surgical Technique (Review).

In this review, we analyzed essential factors affecting precise manual movements in microsurgery described in the medical literature. The search for publications in English and Russian languages was conducted in the PubMed database without limitation by the date of publication. The search was carried out according to the following descriptors: surgical procedures, dexterity, microsurgery, caffeine, alcohol, nicotine, physical exercise, sleep deprivation, posture. Only randomized and cohort studies involving doctors and students with surgical specialties were included in the analysis. We did not include papers in which only psychological (non-motor) aspects were studied. Due to the limited number of publications meeting the inclusion criteria and conflicting results in some of them, the presented review does not allow us to formulate unambiguous conclusions and recommendations. Further studies (deep and fundamental) of endogenous and exogenous factors affecting the microsurgical technique are needed.

[Intraoperative vascular fluorescence in cerebral glioblastomas and vascular histological features]. / Intraoperatsionnaia fluorestsentsiia sosudov v strukture glioblastom golovnogo mozga i ikh gistologicheskaia kharakteristika.

5-ALA intraoperative fluorescence is widely used in surgery of brain tumors for intraoperative demarcation of boundaries and more total resection because 5-ALA metabolites are not accumulated in the intact brain and vascular tissues. Given this fact, it was hypothesized that fluorescence of vessels in the immediate vicinity of a brain tumor may indicate their infiltration by tumor cells as a potential pathway for their dissemination and as a factor for continued tumor growth after surgery and adjuvant therapy. PURPOSE: Identification of fluorescent vessels located near cerebral gliomas, with a histological description of their structure, relationships with the tumor, and potential invasion of the walls by tumor cells. MATERIAL AND METHODS: A prospective cohort study included 14 patients with malignant supratentorial gliomas, aged 20 to 78 years. Five patients were operated on due to continued tumor growth. Two hours before surgery, all patients received 5-ALA orally. During surgery, a microscope (Carl Zeiss OPMI Pentero, Germany) with a fluorescent module (BLUE-400) was used. In all cases, molecular-genetic and immunohistochemical examinations of the tumor material were performed. During surgery, fluorescent vessels, after evaluating their functional significance, were also resected for histological examination. RESULTS: Glioblastoma and anaplastic astrocytoma were verified in 10 and 4 patients, respectively. In 4 out of 10 glioblastoma cases, vessels with homogeneous or fragmentary fluorescent walls were detected in the tumor bed after resection of most of the tumor; in patients with anaplastic astrocytomas, vascular fluorescence was not observed. In the four vascular samples with intraoperatively detected wall fluorescence, tumor invasion into the vascular layers was revealed in all cases. These patients underwent an immunohistochemical examination with monoclonal antibodies to the glial GFAP marker, which clearly identified areas of ingrowth of tumor cells into the vascular wall. CONCLUSION: 5-ALA intraoperative fluorescence is a fundamentally new approach in the rapid diagnosis of tumor-infiltrated blood vessels. Invasion of tumor cells to intact vessels may be a mechanism of tumor progression and dissemination. Additional resection of fluorescent vessels may affect the radicalness of surgical treatment, but requires a mandatory assessment of their functional significance.