A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.

Introduction: Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly. Methods: In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flow cytometry and morphometric analysis of the digital holographic microscopy holograms. Results: Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate. Conclusion: folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.

Extracellular vesicles derived from dendritic cells loaded with VEGF-A siRNA and doxorubicin reduce glioma angiogenesis in vitro.

BACKGROUND: Numerous attempts have been devoted to designing anti-angiogenic agents as a strategy to slow tumor growth and progression. Clinical applications of conventional anti-angiogenic agents face some challenges, e.g., off-target effects for TKIs and also low solid tumor penetration for mAbs. Furthermore, although anti-angiogenic therapy provides a normalization window for better chemo-RT response, in long-term treatments, tumor hypoxia as a result of total removal of VEGF-A by mAbs from the TME or complete blockade of TK receptors induces over-activation of compensatory angiogenic pathways, causing escape. Herein, we investigate the efficacy of si-DOX-DC-EVs to reduce glioma angiogenesis and invasiveness. METHODS: Mature DCs were generated from PBMC and EVs were isolated from the DCs culture media. siRNA and Doxorubicin were loaded into EVs by EP and incubation. Afterward, the uptake of DC-EVs was assessed by flow cytometry, and the subcellular localization of EVs was tested by confocal imaging. Tube formation assay was performed to assess the efficacy of si-DOX-DC-EVs to reduce tumor angiogenesis which was analyzed by DHM. Morphometric analysis of apoptotic cells was performed by DHM and confocal imaging and further, ELISA was performed for hypoxia-related and angiogenic cytokines. The impact of our theranostic system "si-DOX-DC-MVs" on the formation of vascular mimics, colonies, and invasion of C6 cells was checked in vitro. Afterward, orthotropic rat models of glioma were generated and the optimal administration route was selected by in vivo fluorescent analysis. Then, the microvessel density, vimentin expression, and accumulation of immune cells in tumoral tissues were assessed by IHC. Finally, necropsy and autopsy analyses were performed to check the safety of our theranostic agent. RESULTS: DC-EVs loaded with si-DOX-DC-EVs were successfully uptaken by cells with different subcellular trafficking for MVs and exosomes, reduced tumor angiogenesis in DHM analysis, and induced apoptosis in tumoral cells. Moreover, using DHM, we performed a detailed label-free analysis of tip cells which suggested that the tip cells in si-DC-MV treatments lost their geometrical migration capacity to form tube-like structures. Furthermore, the ELISAs performed highlighted that there is a mild overactivation of compensatory Tie2/Ang2 pathway after VEGF-A blockade which confers with severe hypoxia and sustains normal angiogenesis which is the optimal goal of anti-angiogenesis therapy for cancer to avoid resistance.The results of our VM analyses indicated that si-DOX-DC-MVs completely inhibited VM process. Moreover, the invasion, migration, and colony formation of the C6 cells treated with si-DOX-MVs were the least among all treatments. IN was the optimal route of administration. The MVD analyses indicated that si-DOX-DC-MVs reduced the number of tumoral microvessels and normalized vessel morphology. Intense CD8+ T cells were observed near the tumoral vessels in the si-DOX-DC-MVs group and with minimal activation of MT (low Vimentin expression). Necropsy and toxicology results proved that the theranostic system proposed is safe. CONCLUSIONS: DC-EVs loaded with VEGF-A siRNA and Doxorubicin were more potent than BV alone as a multi-disciplinary strategy that combats glioma growth by cytotoxic impacts of DOX and inhibits angiogenesis by VEGF-A siRNAs with excess immunologic benefits from DC-EVs. This next-generation anti-angiogenic agent normalizes tumor vessel density rather than extensively eliminating tumor vessels causing hypoxia and mesenchymal transition.

Efficacy of cell-based immunotherapies on patients with glioma: an umbrella review of systematic reviews and meta-analysis protocol.

INTRODUCTION: Glial brain tumours are highly mortal and are noted as major neurosurgical challenges due to frequent recurrence or progression. Despite standard-of-care treatment for gliomas, the prognosis of patients with higher-grade glial tumours is still poor, and hence empowering antitumour immunity against glioma is a potential future oncological prospect. This review is designed to improve our understanding of the efficacy of cell-based immunotherapies for glioma. METHODS AND ANALYSIS: This systematic review will be performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search of main electronic databases: PubMed/MEDLINE, Scopus, ISI Web of Science EMBASE and ProQuest will be done on original articles, followed by a manual review of review articles. Only records in English and only clinical trials will be encountered for full-text review. All the appropriate studies that encountered the inclusion criteria will be screened, selected and then will undergo data extraction step by two independent authors. For meta-analyses, data heterogeneity for each parameter will be first evaluated by Cochran's Q and I2 statistics. In case of possible heterogeneity, a random-effects meta-analysis will be performed and for homogenous data, fixed-effects models will be selected for reporting the results of the proportional meta-analysis. Bias risk will be assessed through Begg's and Egger's tests and will also be visualised by Funnel plots. ETHICS AND DISSEMINATION: As this study will be a systematic review without human participants' involvement, no ethical registration is required and meta-analysis will be presented at a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42022373297.

Clinical theranostics applications of photo-acoustic imaging as a future prospect for cancer.

Photoacoustic imaging (PAI) of biological tissue has been a fast developing biomedical multi-wave imaging modality, after its introduction in the mid90s. PAI couples laser excitation to acoustic detection. Especially, in recent years its significant advantages in onco-surgery has attracted much attention due to its ability to detect malignant tissues. Monitoring cancer angiogenesis, assessment of blood oxygen saturation, functional brain imaging, evaluation of cortical blood volume, detection of skin/conjunctival melanoma depth, assessment of met-hemoglobin, investigating tumor hypoxia andcancer lymph node metastases are some of its promising applications. Moreover, as a real-time monitoring strategy, PAI allows intraoperative imaging of micro-metastases and residual islands in onco-surgery. Herein, we provide a brief introduction to biophysics and fundamentals of PAI, potential novel endogenous and exogenous contrast agents, and novel techniques to develop engineered and targeted contrast agents with theranostic applications. We also summarize the clinical trial pipelines for PAI. Furthermore, we discuss the potential obstacles and limitation of PAI theranostic agents for further clinical applications and strategies to overcome these hurdles.

Next-Generation Anti-Angiogenic Therapies as a Future Prospect for Glioma Immunotherapy; From Bench to Bedside.

Glioblastoma (grade IV glioma) is the most aggressive histopathological subtype of glial tumors with inordinate microvascular proliferation as one of its key pathological features. Extensive angiogenesis in the tumor microenvironment supplies oxygen and nutrients to tumoral cells; retains their survival under hypoxic conditions; and induces an immunosuppressive microenvironment. Anti-angiogenesis therapy for high-grade gliomas has long been studied as an adjuvant immunotherapy strategy to overcome tumor growth. In the current review, we discussed the underlying molecular mechanisms contributing to glioblastoma aberrant angiogenesis. Further, we discussed clinical applications of monoclonal antibodies, tyrosine kinase inhibitors, and aptamers as three major subgroups of anti-angiogenic immunotherapeutics and their limitations. Moreover, we reviewed clinical and preclinical applications of small interfering RNAs (siRNAs) as the next-generation anti-angiogenic therapeutics and summarized their potential advantages and limitations. siRNAs may serve as next-generation anti-angiogenic therapeutics for glioma. Additionally, application of nanoparticles as a delivery vehicle could increase their selectivity and lower their off-target effects.

Co-expression of cancer stem cell markers, SALL4/ALDH1A1, is associated with tumor aggressiveness and poor survival in patients with serous ovarian carcinoma.

BACKGROUND: Spalt-like transcription factor 4 (SALL4) and aldehyde dehydrogenase1 family member A1 (ALDH1A1) expressing cells have been characterized as possessing stem cell-like properties known as cancer stem cell marker in serous ovarian carcinoma (SOC). METHODS: The association between SALL4 and ALDH1A1 was observed based on literature review and bioinformatics tools. Therefore, this study aimed to investigate the association between the co-expression of SALL4/ALDH1A1 proteins and clinicopathological parameters and their prognostic value in SOC patients using immunohistochemical staining on tissue microarrays (TMAs). Furthermore, benign tumors and normal tissue samples were compared with the expression of the tumor tissue samples. RESULTS: Increased co-expression of SALL4/ALDH1A1 was found to be significantly associated with the advanced FIGO stage (P = 0.047), and distant metastasis (P = 0.028). The results of Kaplan-Meier survival analysis indicated significant differences between disease- specific survival (DSS; P = 0.034) or progression-free survival (PFS; P = 0.018) and the patients with high and low co-expression of SALL4/ALDH1A1, respectively. Furthermore, high level co-expression of SALL4/ALDH1A1 was a significant predictor of worse DSS and PFS in the univariate analysis. The data also indicated that the co-expression of SALL4/ALDH1A1 was an independent prognostic factor affecting PFS. Moreover, the co-expression of SALL4/ALDH1A1 added prognostic values of DSS in patients with SOC who had grade III versus grade I in multivariate analysis. CONCLUSIONS: Our data demonstrated that high co-expression of SALL4/ALDH1A1 was found to be significantly associated with tumor aggressiveness and worse DSS or PFS in SOC patients. Therefore, co-expression of SALL4/ALDH1A1 may serve as a potential prognostic biomarker of cancer progression in these cases.

A comprehensive update to Dendritic Cell therapy for glioma: a systematic review and meta-analysis.

BACKGROUND: Gliomas are major challenges of neuro-oncology due to high mortality. Clinical applications of dendritic cells (DCs) have yielded promising results in the clinical trial pipelines over decades. RESEARCH DESIGN: In this systematic review, we critically discuss the current status, future perspective, and challenges of DC therapy for gliomas . and summarize the study population, blinding, comparators, dosage, treatment regimens, efficacy, and safety issues of the clinical trials published on DC therapy for gliomas and also report the results of our meta-analysis on safety and immunological efficacy of DC therapy for gliomas. RESULTS: The results of our meta-analysis indicated that the most frequent grade I/II adverse event (AE) reported in phase I or phase I/II trials was fatigue (∼16% and 24%). Moreover, in phase II trials, fatigue and cytopenia were the most common AEs (∼9% and 14%). Meanwhile, Grade III/IV AEs were rare . Moreover, our meta-analysis indicated ∼64% CD8+ T cells infiltration into tumor site after DC therapy and also ∼45% IFNγ increase. CONCLUSIONS: DC therapy could serve as a potential immunotherapy for gliomas; however, limitations exist to draw certain conclusions due to diversity of the criteria applied to assess clinical response and limited data on patients' survival.

A systematic update to circulating extracellular vesicles proteome; transcriptome and small RNA-ome as glioma diagnostic, prognostic and treatment-response biomarkers.

Brain gliomas are major neurosurgical challenges due to high mortality and morbidity. Hence, development of novel biomarkers is of great value to plan appropriate treatment strategy. Evaluation of the molecular content of extracellular vesicles (EVs) as novel non-invasive biomarker repertoires can provide a real-time portrait of disease status. This study aims to provide a systematic, comprehensive and critical report of the diagnostic and prognostic significance of EV biomarkers (proteins, DNAs and RNAs) for brain gliomas, discuss their biogenesis and passage through the blood brain barrier, and also highlight the high throughput methods used for EV biomarker discovery; as well as discussing potential limitations of EV isolation and characterization methods as glioma diagnostic, prognostic or treatment response biomarkers. Moreover, we critically appraise the bias risk in the previous studies, discuss the limitations EV biomarker discovery faces to enter neurosurgical practice in the future, and highlight the need for more optimized protocols for EV isolation and biomarker discovery in high throughput studies. The current systematic review was conducted upon PRISMA guidelines [10].

Elevated Lactate and Total Protein Levels in Stereotactic Brain Biopsy Specimen; Potential Biomarkers of Malignancy and Poor Prognosis.

BACKGROUND: Cancer cells bear various metabolic alterations in order to survive and progress. Neoplastic cells rewire their metabolic pathways for fermentation of glucose and production of more lactate. Microenvironment acidification is a common feature of many neoplastic lesions. In other words, most cancer cells produce energy ineffectively, by aerobic glycolysis, considered as "Warburg effect". Mounting on previous evidence, hypoxia also induces tumor stemness, diminished apoptosis, and more invasive behavior as well as angiogenesis. In this study, we aimed to investigate whether more lactate concentration in stereotactic puncture specimen of various brain lesions can be an alarming sign of malignancy and higher grades. The current study aims at providing a rapid prognostic biomarker of higher grades for cystic brain lesions approached stereotactically before the complete pathologic grading report is prepared. METHODS: We investigated the biochemical cyst content of 44 patients with astrocytomas, 8 craniopharyngiomas, 1 oligodendroglioma and 2 cases with metastatic lesions after stereotactic surgery (47 patients were enrolled in the study). Cyst fluid was analyzed for pH, total protein concentration, cytology, and lactate. The association of these parameters was explored relative to tumor behavior (e.g., tumor grade, type, and cyst progression). The current study was conducted at Firoozgar hospital, Tehran, Iran. Patients were followed for any possible progression from 2014 to 2017. RESULTS: The analyses revealed a significant and positive correlation between grade and lactate concentration (P≤0.001); as well as between grade and mean total protein concentration (P = 0.046). This suggests that more lactate and total protein concentration in stereotactic specimen can be an alarming sign of higher grades and poor outcome in astrocytoma cysts. However, craniopharyngiomas; as benign lesions; had significantly lower lactate (P≤0.001) and total protein concentrations (P = 0.018) than astrocytomas. CONCLUSION: Higher total protein and lactate concentrations in the stereotactic biopsy specimens are alarming signs of poor outcome and higher grades in astrocytomas.