Diagnostic Pathology: Open Access
Open Access

Glioma is the most common and fatal type of intracranial tumour; it has an aggressive malignant progression characterised by rapid growth, invasiveness, and a poor prognosis throughout the brain due to many factors [1,2]. Among the many new medicines currently under investigation to target the cell function and signalling pathways of various cancers [3] are Bone Marrow Mesenchymal Stem Cells (BMSCs), which exhibit multidirectional differentiation potential, easy replication and homing, low immunogenicity, easy gene modification, and good immune regulation [4-10]. Studies of BMSCs have attracted great attention [11,12], especially in the development of tumour therapy [13].

BMSCs exhibit inhibitory effects on tumours [14,15], and those carrying exogenous genes strongly inhibit solid tumours [9,16]. Researchers have speculated that BMSCs promote tumour growth and invasion through the multidirectional differentiation of mesenchymal cells and the secretion of cytokines into the tumour microenvironment [10,17]. The self-renewal and in vitro transformation properties of BMSCs are similar to those of tumour cells, and it has been proposed that tumours can be transformed from mesenchymal stem cells [18]. However, BMSCs and cancer cells show significantly different expression of proliferation-associated genes [19], and the bidirectional effects and mechanisms of BMSCs remain controversial [20]. Therefore, it is important to address these issues before widely promoting the clinical use of BMSCs. In this study, we explored whether BMSCs modified with the INF-γ gene could stably express INF-γ in animals with solid tumours and inhibit tumour growth through targeted distribution.

Human glioma U251 cell culture

U251 cells were cultured as described previously [21] at saturated humidity in Roswell Park Memorial Institute (RPMI) 1640 medium, which was changed every 2–3 days.

Primary culture, isolation and identification of BMSCs

Five 8-week-old specific pathogen-free BALB/c mice were purchased from the Experimental Animal Center of the Third Military Medical University, Chongqing, China. The mouse feed was not supplemented with foetal bovine serum in this study. Mice were handled post mortem as described previously [21]. The cell surface molecular markers CD44, CD105, CD34, CD45 and CD11b were detected by flow cytometry [5,22,23].

Development of BALB/c nude mouse models

We obtained and fed 36 BALB/c nude mice (weight, 15 g-20 g; aged 6-7 weeks) as described previously [21]. The experiment was conducted after 1 week of adaptive feeding. All animals were fed according to the feed management specifications for experimental animals using standard pellets provided by the Experimental Animal Center of the Third Military Medical University.

Three mice were randomly selected as a blank group; the remaining mice were used to establish the glioma model as described previously [21]. The weight and growth of the nude mice were observed on the day after tumour inoculation; the tumour dimensions were measured and the volume calculated. Once the subcutaneous tumour had grown to approximately 1 cm in diameter, 30 mice were selected and randomly divided into three groups.

Experimental groups and treatment

The mice were randomly divided into four groups: Negatively control, nude mouse tumour model (model group), lentiviral BMSC transfection with empty vector (BMSC group), and lentiviral BMSC transfection with INF-γ (INF-γ+BMSC group). Prior treatment and screening procedures were performed as described previously [21]. After 1 day, BMSCs transfected with and without INF-γ were injected into mice in the INF-γ+BMSC and BMSC groups, respectively via the tail vein.

INF-γ expression lentiviral vector construction

Target gene fishing, synthesis and purification: The primers used in this study were described previously [21]. The INF-γ overexpression sequence was amplified by Polymerase Chain Reaction (PCR). The target vector was subjected to enzyme digestion and purification.

Target gene and vector-directed connection: The purified PCR product was connected with the linearised vector, and the linked product was transformed into competent bacterial cells. The receptive cells were removed from -80ºC, the centrifuge tube containing the receptive cells was thawed on ice, 10 μL of the connection product was added, and the contents were gently mixed and placed in ice for 30 min. Place centrifuge tube on test tube stand in water bath pre-heated to 42ºC for 60 sec, do not shake centrifuge tube. The centrifuge tube was quickly transferred to the ice bath and the cells were cooled for 5 min. 300 μL LB medium (without antibiotics) was added to each tube, and the tubes were transferred to a 37ºC shaker at 220 rpm for 60 sec to resuscitate bacteria. 100 μL cultured cells were uniformly coated on LB plate containing 50 μg/mL Ampicillin. After the liquid on the plate was absorbed, the plate was inverted into an incubator at 37ºC and cultured for 16 hours. The clonal colonies were selected from the plate, and the positive clones were identified by small plasmid extraction. Four individual, plump colonies were selected from a cultured plate and placed in a test tube containing 5 mL LB medium containing 50 μg/mL Ampicillin. The test tubes were cultured in a bacterial shaker at 37ºC at 250 rpm for 16 h. The cultured bacterial solution was extracted with plasmid small extraction kit. The clones were first identified by enzyme digestion to confirm that the target gene had been directionally linked into the target vector.

Transformation of linker products, positive clone identification, and sequencing: The corresponding bacterial solution of the positive clone was sequenced, and the sequencing result was compared with the target gene sequence. The result indicated successful construction of the target gene expression plasmid vector.

Gene lentivirus packaging and purification: The lentivirus shuttle plasmid and its auxiliary plasmids were prepared and co-transfected into 293T cells, which were cultured in complete culture medium. The medium was replaced at 8 h after transfection, and incubation continued for another 48 h. Cells with a supernatant rich in lentiviral particles were collected. According to the requirements for lentivirus titre in-vivo and in-vitro experiments, the cells were concentrated by density gradient centrifugation to obtain high-titre lentivirus particles, sub packaged for standby, and stored at −80ºC. Fluorescence microscopy or fluorescence-activated cell sorting was used to count the fluorescent cells and calculate the virus titre and dilution ratio.

BMSC gene modification through INF-γ-loaded lentivirus: Once the BMSCs showed adequate growth, we inoculated 104 BMSCs into 24-well plates for transfection. The virus particles were successively warmed to room temperature on a gradient from −80ºC to −20ºC and 4ºC on ice. A pipette was used to transfer exact volumes of the virus solution to the 24-well plate containing prepared culture medium. The virus solution was thus transferred to the target cells and control cells which were incubated overnight under 5% CO₂ at 37ºC after mixing. After 10 h, we replaced the fresh culture medium and continued to culture the transfected BMSCs.

Verification of INF-γ overexpression in BMSCs by quantitative PCR (qPCR): The BMSC and INF-γ+BMSC groups were used to verify the INF-γ transfection effect. After transfection, the media from both groups were discarded and the cells were dissolved with TRIzol reagent. RNA was separated by shaking, followed by standing and centrifugation at 10,000 rpm (all steps at 4ºC). INF-γ was detected by fluorescence qPCR after the separated RNA was dried at room temperature and dissolved in RNase-free water.

All primers were designed using Primer v5.0 software and the gene sequences of IFN-γ and β-actin were obtained from GenBank. No amplification of non-target genes was observed with any of the primers after BLAST verification and none formed a dimer. PCR was performed, the products were electrophoretically separated in 2% agarose gels, and the amplified fragments were observed by gel imaging analysis. The IFN-γ and β-actin genes were amplified by qPCR using a Western Biotechnology fluorescent dye kit under optimised reaction conditions. Following amplification, the fusion and amplification curves were analysed and IFN-γ expression was quantified using the 2^−ΔCt method.

Tumour tissue apoptosis assay

A tumour tissue apoptosis assay was performed as described previously [21]. Cells were stained using a terminal Deoxynucleotidyl Transferase dUTP Nick-End Labelling (TUNEL) apoptosis detection kit, according to the manufacturer’s instructions.

Tumour volume measurement

Tumour volume was measured as described previously [21] using the following formula: Tumour volume = Long diameter × (Short diameter²)/2.

Experimental reagents

RPMI 1640 medium, Foetal Bovine Serum, and Dulbecco’s Modified Eagle Medium were purchased from Gibco (Billings, MT, USA). Dimethyl sulfoxide was purchased from Amresco (Dallas, TX, USA). Reverse transcription reagent, fluorescent qPCR reagent, and SYBR Green I were purchased from Beyotime Biotechnology (Shanghai, China). Other reagents of analytical purity were purchased from domestic reagent suppliers.

Analytical methods

Differences were evaluated using a one-way Analysis of Variance (ANOVA). Data are expressed as means ± Standard Deviation (SD). Student’s t-test was used to evaluate differences between two groups. A repeated measures ANOVA was performed to evaluate differences in multiple factors at different time points between different groups, with time as the dominant effect. Differences were considered significant at P

Primary culture, isolation and identification of BMSCs

BMSCs were cultured in vitro and exhibited adherent growth as described previously [21]. After fluid change, the cells showed clear proliferation and several types of fusiform morphology were observed (Figure 1). Flow cytometry indicated that CD44 (98.01%) and CD105 (96.17%) were overexpressed, whereas CD34 (1.46%), CD45 (1.32%) and CD11b (1.48%) were weakly expressed. Cell morphology and the expression of immune markers on the cell surface were consistent with the literature [5,6,22,23], indicating BMSCs.

Figure 1: Stages of Bone Marrow Mesenchymal Stem Cell (BMSC) primary culture at 2 and 7 days and after passage (10 days). Note: Images were taken under white light (× 100).

Detection of INF-γ overexpression in BMSCs cells by qPCR

Gel electrophoresis of the qPCR products showed clear INF-γ mRNA bands in the INF-γ+BMSC group, but no bands in the BMSC group (Figure 2). Relative INF-γ relative expression was significantly lower in the BMSC group than in the INF-γ+BMSCs group (P <0.05) (Table 1).

Table 1: Relative expression of INF-γ in Bone Marrow Mesenchymal Stem Cells (BMSCs) after transfection.

Figure 2: INF-γ mRNA bands on gel electrophoresis following fluorescence quantitative Polymerase Chain Reaction (qPCR).

Fluorescent INF-γ-transfected BMSC distribution in nude mouse tumours

After immunofluorescence staining, no fluorescent signals were observed in the tumour tissues of mice in the model group under confocal microscopy, whereas fluorescent signals were observed in those of mice in the BMSC and INF-γ+BMSC groups. BMSCs transfected with INF-γ showed a significant homing effect and were concentrated in tumour tissues (Figure 3).

Figure 3: Fluorescence distribution of BMSCs in nude mice observed by confocal microscopy (× 400); (a) Nude mouse tumour model (model group) Lentiviral BMSC transfection groups with (b)empty vector (BMSC group) and (c)INF-γ (INF- γ +BMSC group).

Detection of apoptotic cells by TUNEL staining

TUNEL staining indicated that apoptosis was more prevalent in the BMSC group and the INF-γ+BMSC group than in the model group, whereas the INF-γ+BMSC group showed a clearer distribution of apoptotic cells with brown nuclei (non-apoptotic cell nuclei were counterstained blue with haematoxylin) (Figure 4). For each group, we randomly selected six visual fields in which we counted positive cells (with apoptotic nuclei) and the total number of cells and calculated the ratio of positive cells. Apoptotic cells had higher relative expression levels in the INF-γ+ BMSC group than in the model group (P<0.05) (Table 2).

Table 2: Relative expression of tumour cells in nude mice by Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labelling (TUNEL) staining (x ̅± s).