Establishment of Primary Cell Cultures from Canine Oral Melanomas via Fine-Needle Aspiration: A Novel Tool for Tumorigenesis and Cancer Progression Studies.

Oral melanomas are the most common oral malignancies in dogs and are characterized by an aggressive nature, invasiveness, and poor prognosis. With biological and genetic similarities to human oral melanomas, they serve as a valuable spontaneous comparative model. Primary cell cultures are widely used in human medicine and, more recently, in veterinary medicine to study tumorigenesis, cancer progression, and innovative therapeutic approaches. This study aims to establish two- and three-dimensional primary cell lines from oral canine melanomas using fine-needle aspiration as a minimally invasive sampling method. For this study, samples were collected from six dogs, represented by four primary oral melanomas and five lymph nodal metastases. The cells were digested to obtain single-cell suspensions, seeded in flasks, or processed with Matrigel® to form organoids. The cell cultures were characterized through flow cytometry using antibodies against Melan-A, PNL2, and Sox-10. This technique offers a minimally invasive means to obtain cell samples, particularly beneficial for patients that are ineligible for surgical procedures, and enables the establishment of in vitro models crucial for comparative studies in mucosal melanoma oncology. To the best of our knowledge, this is the first work establishing neoplastic primary cell cultures via fine-needle aspiration in dogs.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

INTRODUCTION: Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. OBJECTIVES: We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. METHODS: To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. RESULTS: Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). CONCLUSIONS: This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Primary microglia cell cultures in translational research: Strengths and limitations.

Microglia are the resident macrophages in the central nervous system, accounting for 10-15% of the cell mass in the brain. Next to their physiological role in development, monitoring neuronal function and the maintenance of homeostasis, microglia are crucial in the brain's immune defense. Brain injury and chronic neurological disorders are associated with neuroinflammation, in which microglia activation is a central element. Microglia acquire a wide spectrum of activation states in the diseased or injured brain, some of which are neurotoxic. The investigation of microglia (patho)physiology and therapeutic interventions targeting neuroinflammation is a substantial challenge. In addition to in vivo approaches, the application of in vitro model systems has gained significant ground and is essential to complement in vivo work. Primary microglia cultures have proved to be a useful tool. Microglia cultures have offered the opportunity to explore the mechanistic, molecular elements of microglia activation, the microglia secretome, and the efficacy of therapeutic treatments against neuroinflammation. As all model systems, primary microglia cultures have distinct strengths and limitations to be weighed when experiments are designed and when data are interpreted. Here, we set out to provide a succinct overview of the advantages and pitfalls of the use of microglia cultures, which instructs the refinement and further development of this technique to remain useful in the toolbox of microglia researchers. Since there is no conclusive therapy to combat neurotoxicity linked to neuroinflammation in acute brain injury or neurodegenerative disorders, these research tools remain essential to explore therapeutic opportunities.

Comparison of permeable cell culture inserts for use in culture of a human in vitro air-liquid interface model system.

In this study, we compared 12 mm cell culture inserts with permeable polyester membranes (0.4 µm pores) from two different manufacturers: CELLTREAT® and Corning®. Physical dimensions and masses of the inserts were found to be very similar between the two brands, with CELLTREAT® inserts having a slightly smaller diameter and growth area (11.91 mm; 1.11 cm2 ) compared to Corning® Transwells® (12 mm; 1.13 cm2 ). We compared cell differentiation outcomes of human nasal epithelial cells (HNECs) at air-liquid interface grown on inserts from the two different manufacturers, including trans-epithelial electrical resistance, ciliary beat frequency, ciliated area, and gene expression. HNECs from three male donors were used for all endpoints. No statistically significant differences were observed between paired cultures grown on different brands of insert. In conclusion, these inserts are comparable for use with airway epithelial cell model systems and likely do not impact cellular differentiation or cell culture quality.

P53-induced GAP-43 Upregulation in Primary Cortical Neurons of Rats.

OBJECTIVES: In this study, we employed an in vitro culturing technique to investigate the impact of p53 on the modulation of growth-associated protein-43 (GAP-43) within the primary cortical neurons of rat specimens. METHODS: (1) Within the first 24 hours after birth, the bilateral cortex was extracted from newborn Wistar rats and primary cortical neurons were cultured and identified. (2) The changes in the mRNA and protein expressions of GAP-43 induced by p53 in rat primary cortical neurons cultured in vitro were identified utilizing real-time polymerase chain reaction and western blot techniques. RESULTS: (1) Lentiviral transfection of p53 within primary cortical neurons of rats elicited elevated levels of both mRNA and protein expressions of GAP-43, consequently culminating in a noteworthy augmentation of p53 expression. (2) The introduction of a p53 inhibitor in rat primary cortical neurons resulted in a reduction in both mRNA and protein expressions of GAP-43. CONCLUSION: Within primary rat cortical neurons, p53 has the potential to prompt an augmentation in both the transcriptional and protein expression levels of the GAP-43 protein.

Isolation, Ex Vivo Expansion, and Lentiviral Transduction of Alveolar Macrophages.

Alveolar macrophages (AM) are resident macrophages of the lung and play important roles in the maintenance of tissue homeostasis as well as host defense. Here, we describe how they can be harvested from murine lungs, expanded in vitro, and transduced with lentiviral vectors.

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

Culturing and Differentiation of Patient-Derived Ectocervical Epithelial Stem Cells Using Air-Liquid Interphase and Matrigel Scaffold.

The ectocervix acts as a multilayered defense barrier, protecting the female reproductive system from external pathogens and supporting fertility and pregnancy. To understand the complex cellular and molecular mechanisms of cervical biology and disease, reliable in vitro models are vital. We present an efficient method to isolate and cultivate epithelial stem cells from ectocervical tissue biopsies. This method combines enzymatic digestion, mechanical dissociation, and selective culturing to obtain pure ectocervical epithelial cells for further investigation. The protocol accommodates both 2D stem cell monolayer and advanced 3D culture systems, such as air-liquid interface and Matrigel scaffolds, using a defined media cocktail, making it highly versatile. The primary ectocervical epithelial cells retain their native characteristics, enabling the exploration of ectocervical epithelial tissue behavior and pathology. This chapter provides step-by-step guidelines for setting up 2D and 3D cultures, facilitating adoption across different laboratories, and advancing cervical biology and disease research.

Inhibition of Oral Fibrinogenesis Through Transforming Growth Factor-Beta/SMAD2/3 Signalling Inhibition Using Mangosteen Pericarp Extract.

Background Chewing areca nuts can result in an oral disorder known as oral submucous fibrosis (OSF), which has the potential to be cancerous. Although it is only beginning to spread to European and the North American continents, it is highly prevalent in Southeast Asia. The probability of malignant transformation from OSF is raised by chewing tobacco use. In the current research, our objective was to assess the potential anti-fibrosis effects and the ability to prevent malignant transformation through the application of mangosteen pericarp extract. Methodology The Ethical Approval-IHEC/SDC/OMED-2101/23/085 from the institution was obtained to conduct this ex vivo study. The cytotoxicity effect of mangosteen pericarp extract on both normal and fibrotic buccal mucosal fibroblasts originating from OSF tissues was tested. Cell proliferation and cell migration by scratch wound healing assay was examined. Dual staining was done to determine the mode of cell death. Additionally, real-time PCR was utilized to measure the expression of TGF-ß/Smad2/3 signalling, α-SMA, and type I collagen gene expression. Results Mangosteen extract exerted higher cytotoxicity of fibrotic buccal mucosal fibroblasts compared to normal cells. Furthermore, mangosteen-receiving cells exhibited downregulation in the expression of the TGF-ß/Smad2 pathway, as well as reduced expression of α-SMA and type I collagen. Conclusion Findings from this study suggest that mangosteen could serve as a promising agent for averting the progression of oral fibrogenesis and halting the malignancy of the oral epithelium in patients with OSF.

Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance.

Intestinal fibroblasts maintain homeostasis and contribute to inflammatory responses and the development of cancer. Intestinal fibroblasts express pattern recognition receptors which can mount an immune response. Since intestinal fibroblasts interact with diverse immune and nonimmune cells, further insights into the biology of intestinal fibroblasts could expand our knowledge of the development, homeostasis, and pathophysiology of the intestine. Here, we describe a simple protocol for the isolation, cultivation, and maintenance of primary fibroblasts from the mouse colon. These cells express α-smooth muscle actin, a characteristic of specialized contractile fibroblasts called myofibroblasts. We also outline the use of these colonic fibroblasts for immunoblotting and immunofluorescence assays with or without stimulation with a growth factor.

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Isolation and Cryopreservation of Mononuclear Cells from Peripheral Blood and Bone Marrow of Blood Cancer Patients.

Peripheral blood and bone marrow aspirates are routinely obtained from blood cancer patients for diagnostic investigations and provide an accessible source of patient-specific cancer cells, as well as non-malignant cells, for research proposes. The simple and reproducible method presented here allows isolation of viable mononuclear cells, including malignant cells, from fresh peripheral blood or bone marrow aspirates using density gradient centrifugation. The cells obtained using the protocol described can be further purified for a variety of cellular, immunological, molecular, and functional assays. In addition, these cells can be cryopreserved and bio-banked for future research studies.

Comparing Cytology Brushes for Optimal Human Nasal Epithelial Cell Collection: Implications for Airway Disease Diagnosis and Research.

Primary nasal epithelial cells and culture models are used as important diagnostic, research and drug development tools for several airway diseases. Various instruments have been used for the collection of human nasal epithelial (HNE) cells but no global consensus yet exists regarding the optimal tool. This study compares the efficiency of two cytology brushes (Olympus (2 mm diameter) and Endoscan (8 mm diameter)) in collecting HNE cells. The study involved two phases, with phase one comparing the yield, morphology and cilia beat frequency (CBF) of cells collected from paediatric participants using each of the two brushes. Phase two compared nasal brushing under general anaesthetic and in the awake state, across a wide age range, via the retrospective audit of the use of the Endoscan brush in 145 participants. Results indicated no significant difference in CBF measurements between the two brushes, suggesting that the choice of brush does not compromise diagnostic accuracy. However, the Endoscan brush collected significantly more total and live cells than the Olympus brush, making it a more efficient option. Importantly, the Endoscan brush is more cost-effective, with a notable price difference between the two brushes.

Extracellular vesicles released by hair follicle and adipose mesenchymal stromal cells induce comparable neuroprotective and anti-inflammatory effects in primary neuronal and microglial cultures.

BACKGROUND AIMS: Despite intensive research, to date, there is no effective treatment for neurodegenerative diseases. Among the different therapeutic approaches, recently, the use of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has gained attention. METHODS: In the present work, we focused on medium/large extracellular vesicles (m/lEVs) derived from hair follicle--derived (HF) MSCs, comparing their potential neuroprotective and anti-inflammatory effect against adipose tissue (AT)-MSC-derived m/lEVs. RESULTS: The obtained m/lEVs were similar in size with comparable expression of surface protein markers. The neuroprotective effect of both HF-m/lEVs and AT-m/lEVs was statistically significant in dopaminergic primary cell cultures, increasing cell viability after the incubation with 6-hidroxydopamine neurotoxin. Moreover, the administration of HF-m/lEVs and AT-m/lEVs counteracted the lipopolysaccharide-induced inflammation in primary microglial cell cultures, decreasing the levels of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1ß. CONCLUSIONS: Taken together, HF-m/lEVs demonstrated comparable potential with that of AT-m/lEVs as multifaceted biopharmaceuticals for neurodegenerative disease treatment.

A modified primary culture method of rat pulmonary vein smooth muscle cells.

BACKGROUND: Although the pressure of pulmonary vein increases before pulmonary artery in pulmonary hypertension due to left heart disease (PH-LHD), only a few studies have assessed pulmonary vein smooth muscle cells (PVSMCs) because of the lack of a simple and feasible isolation method. METHODS: In this study, we introduced a simple method to obtain PVSMCs. Primary pulmonary veins were removed by puncture needle cannula guidance. Then, PVSMCs were cultured by the tissue explant method and purified by the differential adhesion method. The cells were characterized by hematoxylin-eosin (HE) staining, immunohistochemistry, western blotting, and immunofluorescence to observe the morphology and verify the expression of alpha-smooth muscle actin (α-SMA). RESULTS: The HE staining results showed that the pulmonary vein media was thinner than the pulmonary artery, the intima and adventitia of the pulmonary vein were removed by this method, and the obtained cells with good activity exhibited morphological characteristics of smooth muscle cells. In addition, higher α-SMA expression was observed in the cells obtained by our isolation method than in the traditional method. CONCLUSION: This study established a simple and feasible method to isolate and culture PVSMCs that might facilitate the cytological experiments for PH-LHD.

Comparison of biological behavior of lacrimal gland adenoid cystic carcinoma with high-grade transformation cells.

AIM: To evaluate the differences between human lacrimal gland adenoid cystic carcinoma with high-grade transformation (LACC-HGT) primary cells cultured by high-grade transformation tissue and non-high-grade transformation (non-HGT) primary cells cultured by non-high-grade transformation tissue in proliferation, metastasis, drug susceptibility, and genes. METHODS: LACC-HGT primary cells were established by tissue block culture, and the 4th to 10th generation primary cells were selected as research objects. The cells were preliminarily identified by immunofluorescent staining. The differences between non-HGT and LACC-HGT primary cells in terms of proliferation, metastasis, and drug susceptibility were compared by cell counting kit-8 (CCK-8) assay, wound healing, and drug sensitivity experiments. Differentially expressed genes were screened using mRNA array. Gene expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: LACC-HGT primary cells were successfully cultured by tissue block culture. Immunofluorescence staining results showed that cytokeratin (CK) and CK7 expression levels were positive in LACC-HGT primary cells. CCK-8 results showed that the proliferation ability of LACC-HGT cells was significantly higher than that of non-HGT cells. Wound healing experiment showed that the migration ability of LACC-HGT cells was significantly higher than that of non-HGT cells. LACC-HGT cells were also less sensitive to cisplatin and paclitaxel than non-HGT cells. Compared with non-HGT cells, 9566 differentially expressed genes were found in LACC-HGT primary cells, of which 5162 were up-regulated and 4404 were down-regulated. The expression of N-acetylneuraminate pyruvate lyase (NPL), MARVEL domain containing 3 (MARVELD3), syntabulin (SYBU), and allograft inflammatory factor 1 (AIF1) was higher in LACC-HGT cells than in non-HGT cells, whereas that of periostin (POSTN) was lower. CONCLUSION: LACC-HGT primary cells have faster proliferation, stronger migration ability, and poorer sensitivity to chemotherapy drugs than non-HGT primary cells. The expression of mRNAs in non-HGT and LACC-HGT primary cells are significantly different. These features are speculated to be the reasons why high-grade transformation tissues exhibit higher malignant degree and poorer prognosis than their counterparts.

Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Establishment of a diverse head and neck squamous cancer cell bank using conditional reprogramming culture methods.

Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from the oral cavity, 4/16 were derived from the oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papillomavirus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, reverse transcription polymerase chain reaction (RT-PCR) of HPV16/18-specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of four oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity.

Isolation of Cells and Exosomes from Glioblastoma Tissue to Investigate the Effects of Ascorbic Acid on the c-Myc, HIF-1α, and Lnc-SNHG16 Genes.

Glioblastoma multiforme (GBM) is incurable with routine treatments. Ascorbic acid (Asc) has antioxidant and anti-cancer properties. However, its specific anti-cancer mechanisms are only partially understood. In this study, the effect of Asc on the c-Myc, HIF-1α, and lnc-SNHG16 genes in GBM cells and their exosomes was investigated. Cells isolated from the tissue were characterized by the immunocytochemistry method (GFAP+). The cell-doubling time was determined, and FBS-free medium supplemented with Asc (5 mM) was added to the cells. The extracted exosomes in the cell culture medium were scanned by electron microscopy, Zetasizer, and BCA assay. The expression of lnc-SNHG16 in the exosomes and c-Myc and HIF-1α in the treated and control cells was evaluated by real-time PCR. The interactions between Asc and the c-Myc and HIF-1α proteins were studied using the molecular docking method. The cells showed 90-100% GFAP+ in passage 4, with a cell-doubling time of 4.8 days. Exosomal vesicles measuring 98.25-105.9 were observed. Zetasizer results showed a sharp pick at 90 nm. Protein quantitation showed 3.812 µg/ml protein in the exosomes. Lnc-SNHG16 expression was reduced (P = 0.041), and c-Myc was upregulated (P = 0.002). The expression of HIF-1α was not significant in the treated cells. Also, Asc was able to interact and affect c-Myc and HIF-1α. Asc exerts its effect by reducing lnc-SNHG16 expression in exosomes, upregulating c-Myc in GBM cells, and interacting with HIF-1α and c-Myc. Further research is necessary to achieve a full understanding of these findings.