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.

[High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera].

The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNt and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.

High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera.

The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNT and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.

[Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures].

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out ß2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1^(+) overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^(-/-) or B2M^(-/-) knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^(-/-) has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1^(+)-CISH^(-/-) and YT-VAV1^(+)B2M^(-/-) lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1^(+) line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

[Infections and inflammation in the development of stroke]. / Infektsii i vospalenie v razvitii insul'ta.

The review systematizes data on the role of infectious diseases and systemic inflammation in the pathogenesis of stroke. Various risk factors for stroke associated with pro-inflammatory reactions and their contribution to the pathogenesis of cerebrovascular pathology are analyzed. The interaction of systemic inflammation with hemostasis disturbances and clots formation, activation of autoreactive clones of cytotoxic lymphocytes, the progression of endothelial damage, and other processes is shown. Along with infection, these factors increase the risk of stroke. The key mechanisms of the pathogenesis from the development of acute or chronic inflammation to the preconditions of stroke are presented. The mechanisms of the acting of the infectious process as a trigger factor and/or medium-term or long-term risk factors of stroke are described. A separate section is devoted to the mechanisms of developing cerebrovascular diseases after COVID-19. Identifying an increased risk of stroke due to infection can be of great preventive value. Understanding of this risk by specialists followed by correction of drug therapy and rehabilitation measures can reduce the incidence of cerebrovascular complications in infectious patients.

Bioluminescent Study of the Distribution of High-Molecular-Weight Protein Fraction of Cellex Daily Preparation in the Brain after Intranasal Administation.

Permeability of the blood-brain barrier for protein fractions 50-100 kDa (PF50-100) of Cellex Daily preparation labeled with fluorescent tracer FITC and non-conjugated FITC were compared after intranasal administration of the preparations to healthy rats. Fluorimetrical analysis of the serum and cerebrospinal fluid samples showed that Cellex Daily PF50-100-FITC administered intranasally penetrated into the blood and cerebrospinal fluid with maximum accumulation in 2 h after administration and persists in the circulation for 24 h probably due to binding with plasma proteins. The differences in the kinetic profile of PF50-100-FITC and free FITC indirectly suggest that the major part of the preparation is not degraded within 24 h and FITC is probably not cleaved from the protein components of the preparation. In vivo fluorescence analysis showed significant fluorescent signal in the olfactory bulbs in 6 h after intranasal administration; hence, the preparation administered via this route can bypass the blood-brain barrier. Scanning laser confocal microscopy of rat brain sections confirmed penetration of the high-molecular weight protein fraction PF50-100-FITC into CNS structures. The most pronounced accumulation of the labeled drug was observed in the olfactory bulb in 6 and 12 h after administration. In contrast to free FITC administered in the control group, significant accumulation of PF50-100-FITC in the olfactory cortex and frontal cortex neurons with functionally active nuclei was observed in 6, 12 and 24 h after intranasal administration.

Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer.

Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-µm metastases in multiple organs and bones at the early stage of their development. After 3-4 weeks, 4T1 generated 11.4 ± 2.1, 4T1luc2D6, 4.5 ± 0.6; and 4T1luc2, <1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-γ response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.

Mono- and Combined Therapy of Metastasizing Breast Carcinoma 4T1 with Zoledronic Acid and Doxorubicin.

The efficiency of monotherapy with zoledronic acid (Resorba), doxorubicin, and their combination was studied on the model of metastasizing breast carcinoma in BALB/c mice. Doxorubicin monotherapy was accompanied by a significant increase in median survival up to 57 days (vs. 34 and 35 days in control groups); 27% animals survived for 90 days (duration of the study). Bioluminescence area of the primary tumor significantly decreased on days 21 and 28; the total number of visceral metastases also decreased according to magnetic-resonance imaging data. Resorba monotherapy produced no general toxic effect, the median survival increased to 64 days, and 90-day survival was 33%. Imaging techniques (magnetic-resonance imaging, microtomography, bioluminescent analysis) showed that Resorba delayed the development of the primary tumor (regression of luminescence area on days 21 and 28, regression of standardized bioluminescence intensity on day 28) and significantly reduced the number of visceral metastases in comparison with the control. Combination therapy was less effective than monotherapy with the same medications. Median survival was 55 days, 90-day survival was 13%, but magnetic-resonance imaging and bioluminescence analysis after combination therapy also showed delayed growth of the primary tumor and reduced number of visceral metastases. Microtomography revealed bone metastases in ~30% animals of the control group; in experimental groups, no bone metastases were found. The experiment with periosteal (distal epiphysis of the femur) injection of 4T1-Luc2 tumor cells demonstrated pronounced selective effectiveness of Resorba in relation to bone metastases. Monotherapy with Resorba can prevent the development of not only bone, but also visceral metastases of breast cancer.

[Blood-brain barrier permeability in healthy rats and rats with experimental C6 glioma after fractionated radiotherapy of the brain].

OBJECTIVE: To evaluate the effect of fractionated radiotherapy on permeability of the blood-brain barrier in healthy rats and rats with C6 glioma in vivo. MATERIAL AND METHODS: An increase in BBB permeability in C6 glioma was assessed by dynamic MRI monitoring (glioma size before and after radiation therapy in combination with immunotherapy, n=30) and confocal microscopy (fluorescence imaging of tumor invasion boundaries in a dose-dependent experiment for the amount of injected antibodies). In healthy rats, BBB permeability to macromolecular substances (MMS) was assessed by ELISA (n=23, 192 plasma samples) and confocal microscopy (n=7). RESULTS: It was shown that BBB permeability to biological macromolecules in blood-brain and brain-blood directions was increased after fractionated radiotherapy. CONCLUSION: Drug delivery to the brain can be improved using therapeutic doses of radiation treatment that affects the BBB and minimizes the risk of serious side effects that are often associated with the drug dose.

Modeling and integral X-ray, optical, and MRI visualization of multiorgan metastases of orthotopic 4T1 breast carcinoma in BALB/c mice.

A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.

Expression of VEGF, GFAP, and BDNF genes in the brain of rats after fractionated γ-irradiation according to different protocols.

The expression of VEGF, GFAP, and BDNF genes in the nervous tissue changed on weeks 4, 8, and 12 after fractionated irradiation of the brain according to different protocols in a fixed total dose of 36 Gy. The expression of VEGF gene decreased in the prefrontal cortex and hippocampus after 4 and 8 weeks. After week 12, the expression of VEGF normalized in the prefrontal cortex and remained low in the hippocampus. The expression of GFAP gene was maximum in the prefrontal cortex after week 4 and returned to normal in week 12. In the hippocampus, the expression of GFAP was low only after week 12. The expression of BDNF gene was reduced only during the week 8 and this decrease was directly proportional to the single dose. Hence, fractionated γ-irradiation with fixed total dose differently modified gene expression in the nervous tissue.

Treatment of poorly differentiated glioma using a combination of monoclonal antibodies to extracellular connexin-43 fragment, temozolomide, and radiotherapy.

Antitumor efficiencies of monoclonal antibodies to connexin-43 second extracellular loop (MAbE2Cx43), temozolomide, and fractionated γ-irradiation in the monotherapy mode and in several optimized combinations were studied in Wistar rats with induced C6 glioma. The survival of animals with glioma and the dynamics of intracerebral tumor development were evaluated by MRT. Temozolomide monotherapy (200 mg/m(2)) and isolated radiotherapy in a total dose of 36 Gy shifted the survival median from 28 days (no therapy) to 34 and 38 days, respectively; 100% animals died under conditions of temozolomide monotherapy and radiotherapy. Monotherapy with MAbE2Cx43 in a dose of 5 mg/kg led to significant regression of the tumor (according to MRT data), cure of 19.23% animals with glioma, and prolongation of the survival median to 39.5 days after tumor implantation. Combined therapy with MAbE2Cx43 and temozolomide completely abolished the antitumor effect (survival median 29 days). Treatment with MAbE2Cx43 in combination with radiotherapy was associated with mutual boosting of the therapeutic efficiencies, leading to a significant inhibition of tumor development and prolongation of the survival median to 60 days. The mechanism of tumorsuppressive activity of the antibodies could be due to connexon blockade in Cx43-positive glioma cells in the peritumor invasion zone. Higher efficiency of combined therapy was presumably due to the increase in blood-brain barrier permeability for antibodies after irradiation of the brain and to additional inhibitory effect of antibodies towards radioresistant migrating glioma cells. The results suggested that MAbE2Cx43 could be effective as the first-line drug in combined therapy for poorly differentiated gliomas.

Targeted delivery of cisplatin by сonnexin 43 vector nanogels to the focus of experimental glioma C6.

The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors.

Antitumor effects of monoclonal antibodies to connexin 43 extracellular fragment in induced low-differentiated glioma.

We studied the effect of intravenous administration of monoclonal antibodies to the second extracellular loop of connexin 43 (MAbE2Cx43) on the dynamics of glioma growth and survival of experimental animals. Morphometric analysis of magnetic resonance imaging data showed that weekly intravenous administration of MAbE2Cx43 in a dose of 5 mg/kg significantly reduced glioma volume starting from day 21 after tumor implantation. By day 29, the mean volume of glioma in the experimental group (therapy with specific antibodies) was 2-fold lower than in controls. Deceleration of glioma growth in rats receiving MAbE2Cx43 was accompanied by a significant prolongation of rat lifespan (according to Kaplan-Meier test) and even led to complete recovery without delayed relapses in 19.23% animals. The mechanism of tumor-suppressing effects of antibodies can be related to inhibition of specific functions of connexin 43 in glioma cells in the peritumoral zone.

Neural progenitor and hemopoietic stem cells inhibit the growth of low-differentiated glioma.

The effects of neural progenitor and hemopoietic stem cells on C6 glioma cells were studied in in vivo and in vitro experiments. Considerable inhibition of proliferation during co-culturing of glioma cells with neural progenitor cells was revealed by quantitative MTT test and bromodeoxyuridine incorporation test. Labeled neural progenitor and hemopoietic stem cells implanted into the focus of experimental cerebral glioma C6 survive in the brain of experimental animals for at least 7 days, migrate with glioma cells, and accumulate in the peritumoral space. Under these conditions, neural progenitor cells differentiate with the formation of long processes. Morphometric analysis of glioma cells showed that implantation of neural progenitor and hemopoietic stem cells is accompanied by considerable inhibition of the growth of experimental glioma C6 in comparison with the control. The mechanisms of tumor-suppressive effects of neural and hemopoietic stem cells require further investigation.

Immunochemical analysis of glial fibrillary acidic protein as a tool to assess astroglial reaction in experimental C6 glioma.

In experiments on Wistar rats with experimental C6 glioma, the immunohistochemical features of the astroglial reaction over 30 days after implantation were characterized. The formation of a glial border consisting of GFAP-positive reactive astrocytes at the periphery of C6 glioma was observed on postimplantation day 3 and until the death of the experimental animals. Reactive astrocytes encompassed not only the primary gliomal focus, but all tumor invasion foci. Quantitative assessment of astroglial reaction around glioma was carried out with immunofluorescent assay of glial fibrillary acidic protein (GFAP) on cerebral sections. The size of glioma and necrotic foci were analyzed morphometrically in parallel with enzyme immunoassay of serum GFAP. A correlation between morphometric indices of glioma and serum level of GFAP was found. It was concluded that serum concentration of GFAP correlated with the size of intracranial glioma, necrotic foci, and most strongly with the degree of reactive astrogliosis. Monitoring of the level of serum GFAP can serve as an additional diagnostic index reporting the state of intracranial glioma.

Isolation of extracellular recombinant fragment of rat connexin-43.

Analysis of membrane topology of connexin-43 (Cx-43) made it possible to determine one of its extracellular fragments (E2): Q173-1208. The nucleotide sequence of this fragment was cloned into pCBDQ and pHPML vectors containing the sequences of calmodulin-binding domain (CBD) and HPML-domain of Ca-ATPase of human hPMCA4b cells plasma membrane. This yields two chimeric proteins with N-terminal 6-histidine motif containing the extracellular fragment Cx43 E2 and one of hPMCA4b domains (Cx43-CBD and Cx43-HPML). The latter were inserted into the recombinant polypeptide to improve solubility and enhance immunogenicity of the product. Affinity-purified on Ni-NTA agarose recombinant Cx43-CBD was used for immunization of mice and obtaining of monoclonal antibodies. Primary selection of clones was carried out by solid-phase IEA with immobilized Cx43-HPML and by immunoblotting with Cx43-HPML. The positive clones were tested immunohistochemically on rat brain sections. This two-stage testing made it possible to select two hybridomas, which produced monoclonal antibodies to Cx43 in native conformation. The resultant antibodies can be used in vitro and in vivo for immunophenotyping of various Cx43-positive cells.

Immunofluorescent analysis of connexin-43 using monoclonal antibodies to its extracellular domain.

Immunofluorescent analysis of connexin-43 was carried out on preparations of fixed and living cultures of rat and human glioma cells, HEK293 cells, and frozen sections of the rat brain with experimental glioma using monoclonal antibodies to recombinant extracellular fragment of connexin-43 (E2 second extracellular loop). These monoclonal antibodies visualized membrane and cytoplasmic pools of connexin-43 in preparations fixed with paraformaldehyde. Incubation of monoclonal antibodies to E2 extracellular loop with living cells led to visualization of only connexin hemichannels on cell membranes. No immunofluorescence characteristic of dimer connexons, organizing the gap junction, was detected. This fact indicates that antibodies to connexin-43 extracellular loop E2, obtained in our study, specifically react with target antigen solely at the stage of connexon presentation on the membrane in the form of hemichannels. These monoclonal antibodies can be used for immunophenotyping and sorting of connexin-43-positive cells in vitro and as the guide molecules in addressed delivery of diagnostic preparations and drugs to glioma cells in vivo.

Modeling and immunohistochemical analysis of C6 glioma in vivo.

A reproducible in vivo model of C6 glioma was developed in Wistar rats. Analysis of histological preparations showed similar morphology of rat C6 glioma and human glioblastoma. The formation of a glial border at the periphery of the glioma, consisting of GFAP-positive reactive astrocytes, was shown by the immunohistochemical method. The border appeared on day 8 after implantation, astrogliosis was observed until animal death (day 28). Reactive astrocytes with branched processes surrounded not only the primary glioma focus, but also all sites of tumor invasion in the nervous tissue. Expression of EBA (blood-brain barrier marker) was disturbed and synthesis of AMVB1 (endothelial antigen) increased in neoplastic endotheliocytes, which suggested pronounced functional restructuring of the blood-tumor barrier in comparison with the blood-brain barrier. The phenomenon of predominant expression of GFAP and AMVB in the tumor tissue can be used for the development of systems for targeted drug transport into the tumor by means of appropriate antibodies.