CB2R activation enhances tumor-associated macrophages-mediated phagocytosis of glioma cell

2.1. Animals

Male C57BL/6 mice, aged 4–6 weeks, were obtained from the Experimental Animal Centre at the Third Military Medical University and were maintained in the Laboratory Animal Centre of Southwest Hospital (Gaotanyan Street No.30, Shapingba District, Chongqing, China). In vivo experiments did not involve randomization. The mice were housed in a temperature-controlled environment devoid of particular pathogens, with a standard 12-h light/dark cycle and unrestricted access to food and water. The experimental protocols follow the Animal Research: Reporting in Vivo Experiments criteria. The Ethics Committee of the Third Military Medical University approved the experimental protocols (AMUWEC20222930), which were conducted following the Guide for the Care and Use of Laboratory Animals.

2.2. Intracranial GBM xenografts and treatment

Professor Haofei Liu (Third Military Medical University, Chongqing, China) generously donated GL261 cells, a murine GBM cell line generated from C57BL/6 mice [25]. The GL261 cells originated from the National Cancer Institute in Frederick, Maryland [26]. To maintain cellular proliferation at 37 °C, 10 % fetal bovine serum (FBS, Gibco), was added to DMEM/F12 (HyClone). GL261 cells were transfected with pLV-GFP-luciferase (Luc) lentivirus, as previously described; flow cytometry was used to select GFP-positive GL261-GFP-Luc cells to generate a Luc-stable cell line [25]. Ketamine (50 mg/kg) and xylazine (5 mg/kg) were administered intraperitoneally into C57BL/6 to induce anesthesia in the GL261 tumor model. Following shaving, mouse heads were positioned within a stereotaxic frame. Following alcohol and betadine sterilization of the scalp, a midline scalp incision measuring 1 cm was performed to gain access to the cranium. Following that, the periosteum was extracted using 4 % H₂O₂. Next, starting from the bregma, a burr incision was performed 2 mm laterally and 0.8 mm anteriorly to the midline. A 10 μL syringe bearing a 33 G needle was withdrawn 0.5 mm from a depth of 3 mm to create a reservoir. Using a micro infusion pump, 10⁴ tumor cells in a volume of 3 μL were administered at a rate of 0.5 μL/min. After being in place for 5 min, the syringe was removed. Bone wax was used to fill the burr hole, and the skin was cleaned and conglutinated. At least 6 mice in each group were housed in a warm cage until they recovered completely.

On the seventh day following tumor implantation, mice were assigned to either the control or treatment group according to the bioluminescent signal of the initial tumor burden. To determine the effect of CB2R manipulation on tumor growth with or without TMZ treatment, mice were treated with vehicle, TMZ (5 mg/kg, i.p., Selleckchem, S1237), AM630 (1.0 mg/kg, Tocris Bioscience, 1120), JWH133 (1.0 mg/kg, Tocris Bioscience, 1343), or the combination of TMZ and JWH133. JWH133 and AM630 were intraperitoneally injected every day from day 3 after tumor cell implantation for 10 days. Five TMZ intraperitoneal injections were administered every 2 days beginning on the third day following tumor cell implantation [26]. To further verify whether CB2R enhances phagocytosis by up-regulating CD36 on tumor-associated macrophages, the xenografted mice were intraperitoneally injected with the sulfo-N-succinimidyl oleate (SSO, 15 mg/kg, MedChemExpress, HY-112847A), an irreversible inhibitor of the CD36 [27,28], every day from day 3 after tumor cell implantation for 10 days. Six mice per treatment group were randomly selected for live imaging, immunofluorescence, flow cytometry, and western blot. There are different batches of samples for above experiments and examined at day 14 after implantation. The sample size was at least nine mice per group for animal survival between groups.

2.3. Immunofluorescence

The protocols for immunofluorescence procedures followed those outlined by Yang et al. [29]. Briefly, the mice in each group were deeply anesthetized and perfused transcardially with 0.01M PBS followed by 4 % paraformaldehyde in 0.01M PBS. The whole brains were isolated, post-fixed in 4 % paraformaldehyde for 24–48 h, and then stored in 30 % sucrose in 0.01M PBS solution for 48 h for cryoprotection. After embedding and freezing with optimal cutting temperature compound (OCT), brains were sectioned into 30 μm-thick using a cryostat (Leica). The cryostat brain slices were treated with primary antibodies in 1 % bovine serum albumin (BSA), then the slices were incubated with mouse anti-CD11b (ab133357, 1:500, Abcam, Cambridge, MA) and chicken anti-GFP (ab13970, 1:1,000, Abcam) and maintained at 4 °C for the night. Following incubation, sections were washed with 0.01 M phosphate-buffered saline and subsequently labeled with Alexa Fluor Cy3 and 488 conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:500 for 2 h at room temperature (RT). After that, sections were embedded in Vectashield mounting solution (Vector Laboratories, Burlingame, CA) and counterstained with 4′,6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Santa Cruz, CA) before being photographed using a Zeiss confocal microscope (LSM780; Zeiss, Thornwood, NY). For quantification, the phagocytosis cell percentage was determined in five randomly chosen fields for each of the at least 6 mice per group.

2.4. Flow cytometry analyses

In the flow cytometry experiment, the mice in each group underwent deep anesthesia and transcardial perfusion with 0.01M PBS. The whole brains were dissected under a stereo fluorescence microscope (Nikon) to isolate the GFP-positive tissues with a sterile scalpel. The collected GFP-positive tissues were dissected into small pieces using scissors. These pieces were then placed in C-Tubes (Miltenyi Biotec, 130-096-334) containing a digestion cocktail (11088882001, Roche, Indianapolis, IN; MO D7291-2 MG, Sigma-Aldrich, St. Louis) containing 1 mg/mL collagenase D and 30 mg/mL DNase I (11088882001, Roche) in complete RPMI 1640 medium (10 % FBS). The temperature was maintained at 37 °C for 15 min. Following that, the tissue was gently dissociated utilizing a MACS and subsequently incubated at 37 °C for an additional 15 min. The homogenate was subsequently filtered through a 70 μm strainer and centrifuged at 4 °C for 10 min at 500×g. The resulting pellet was resuspended in RPMI-1640 medium supplemented with 10 % FBS. Following this, an immune cell enrichment step was performed using density gradient centrifugation with 30 % and 70 % Percoll (Fisher Scientific, 10607095) in RPMI 1640 medium containing 10 % FBS. Upon centrifugation for 20 min at 20 °C and 2000×g (without using any force during acceleration or deceleration), the immune cells-containing middle transparent layer was carefully extracted.

Singular-cell suspensions were generated from complete GL261-GFP-Luc allografts as previously documented [25] to quantify the proportion of TAMs exhibiting phagocytosis in the tumor tissue of the GBM model. To inhibit non-specific and Fc-mediated binding, the cells were incubated in ice with anti-CD16/CD32 (553141; BD Biosciences, San Jose, CA) antibodies. Following a 10-min incubation period, three washes and an additional 60 min of dark incubation were required for the GL261-GFP-Luc allograft single-cell suspensions to be treated with APC anti-mouse CD11b antibody (101211, 1:1000; Biolegend, San Diego, CA). Isotype controls were employed for every staining attempt. The data acquisition process was conducted on BDFACSAria II, and FlowJo X 10.0.7 (TreeStar, Ashland, OR) was utilized to analyze and present the data.

2.5. Living imaging

To measure tumor growth, tumor-bearing mice were anesthetized with isoflurane 14 days following tumor implantation and administered an intraperitoneal injection of D-Luciferin (150 mg/kg body weight; Cat# P1043; Promega, Madison, WI). After 10 min of being moved to the in vivo imaging system (IVIS) 100 imaging equipment (Caliper Life Sciences, Alameda, CA), the animals’ luminescence was detected. Following this, the data were analyzed utilizing the Living Image 2.5 software (Coronar Life Sciences).

2.6. Western blotting

The flow cytometry-sorted CD11b-positive TAMs of different groups were lysed in accordance with a process published in a prior work [6]. Specifically, ice-cold RIPA buffer (Sigma-Aldrich) was utilized, which had been enriched with a protease inhibitor cocktail (Roche). The levels of protein were quantitatively assessed through the Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China). Following that, polyvinylidene fluoride membranes were utilized to transfer the separated samples, each of which contained 20 mg of protein, via 10 % SDS-PAGE (Roche). Primary antibodies against CD36 (rabbit, ab252923, 1:1000; Abcam) and GAPDH (mouse, ab8245, 1:1000; Abcam) were incubated at 4 °C overnight after the membranes had been incubated at RT for 2 h in Tris-buffered saline with 0.1 % Tween 20 detergent containing BSA and 0.05 % Tween 20. Following this, the membranes were incubated at RT for 1 h with an HRP-linked secondary antibody The protein bands were identified utilizing the WesternBright ECL kit (Advansta, Menlo Park, CA) in conjunction with the ChemiDocTM XRS C imaging system (Bio-Rad, Berkeley, CA; Densitometry was performed on each of the membranes utilizing the Image LabTM software (Bio-Rad).

2.7. Statistical analysis

Statistical analyses in this study were conducted using GraphPad Prism 8.0 and SPSS 18.0 software. The Kolmogorov-Smirnov test was utilized to evaluate whether the data were normally distributed. Comparisons between 2 groups were analyzed using 2-tailed Student t tests. Tukey’s post hoc test was applied after one-way ANOVA (one factor) was utilized to examine intergroup comparisons. Survival rates were analyzed using the Kaplan-Meier (K-M) method, and the differences between groups were compared using the log-rank test. The format of mean ± SEM was used to express the data, and a P value < 0.05 was deemed to demonstrate statistical significance.

3.1. CB2R activation impairs GBM growth and improves the survival of xenografted mice

We created GBM xenografts from Luc-expressing GL261 cells to determine whether CB2R activation affects the progression of GBM in vivo. Mice were treated with JWH133 (a selective CB2R agonist), AM630 (a selective CB2R antagonist), or the vehicle 3 days after GL261-Luc cells were implanted. On day 14 post-implantation, the tumor growth was tracked using an IVIS and a bioluminescence signal. In comparison to the Control group, JWH133 therapy inhibited GBM growth, according to bioluminescence analysis (Fig. 1A–B, P < 0.01). On the contrary, mice treated with AM630 experienced a considerably quicker progression of GBM in comparison to the Control group (Fig. 1A–B, P < 0.0001). The K-M survival analysis showed that the administration of JWH133 produced a survival advantage over the Control group in line with the reported consequences on tumor growth (Fig. 1C, P = 0.0385). In contrast, survival was significantly reduced in mice treated with AM630 relative to the Control group (Fig. 1C, P = 0.0149). Overall, these findings demonstrated that CB2R activation impedes GBM growth and enhances the survival of xenografted mice.

3.2. CB2R activation promotes TAMs-mediated phagocytosis of glioma cells

To assess whether CB2R activation enhances TAMs-mediated phagocytosis of glioma cells, we administered JWH133 and AM630 to xenografted mice bearing GL261-GFP cell line tumors for 10 days. Histologically, the CD11b, labeled with RFP, served as a marker for TAMs. Consequently, phagocytes were identified by the presence of red protrusions containing GFP-positive cells. This method facilitated the characterization of TAMs-mediated phagocytosis within the experimental model. Our findings demonstrated that treatment with JWH133 significantly increased the phagocytosis of GFP-positive glioma cells by TAMs in comparison to the Control group (Fig. 2 A and C, P < 0.01). Conversely, AM630 treatment led to a notable inhibition of TAMs-mediated phagocytosis compared to the Control group (Fig. 2 A and C, P < 0.05). Following the results of the flow-based phagocytosis assay, treatment with JWH133 significantly increased phagocytosis mediated by TAMs in comparison to the Control group (Fig. 2 B and D, P < 0.0001), whereas treatment with AM630 substantially decreased this process in comparison to the Control group (Fig. 2 B and D, P < 0.05). Thus, CB2R activation is a crucial mediator of the phagocytic activity of TAMs, according to the collective findings presented herein.

3.3. CB2R activation promotes TAMs-mediated phagocytosis of glioma cells via increasing the expression of CD36 in TAMs

CD36, which belongs to the class B scavenger receptor family, is essential for the phagocytic activity of macrophages, monocytes, and microglia, among other immune cells. Given its role in phagocytosis, CD36 has garnered significant interest in studies focused on inflammation, atherosclerosis, and neurodegenerative diseases, among other areas [30]. To investigate whether CD36 mediates the CB2R-induced phagocytic activity of TAMs in glioma, we sorted CD11b-positive TAMs using flow cytometry and examined the expression of CD36 in each group. According to Western blotting analysis findings, TAMs treated with JWH133 had considerably higher CD36 expression than the Control group (Fig. 3A–B, P < 0.05). However, AM630 treatment dramatically decreased CD36 expression in TAMs as compared to the Control group (Fig. 3A–B, P < 0.001). What’s more, to verify whether CB2R enhances phagocytosis by increasing CD36 levels on tumor-associated macrophages, we treated xenografted mice with SSO, an irreversible CD36 inhibitor [27]. Remarkably, immunofluorescence and flow cytometry results demonstrate that SSO treatment reversed the CB2R activation-induced enhancement of phagocytosis by TAMs (Fig. 3C–F, P < 0.01). These findings suggest that CB2R activation increases CD36 expression, which in turn promotes the phagocytic activity of TAMs. This sheds light on a potential mechanism underlying the observed effects of CB2R modulation on TAMs-mediated phagocytosis in glioma.

3.4. JWH133 enhances the therapeutic efficacy of TMZ

Due to resistance in certain patients, TMZ, a primary chemotherapy for GBM that is well-known for its capacity to cross the blood-tumor barrier and cause cytotoxicity by breaking DNA double strands, is not always effective. This resistance is often correlated with mutations in isocitrate dehydrogenase 1 and elevated levels of O6-methyl guanine-DNA methyltransferase promoter methylation [31]. A synergistic approach, combining TMZ with immunotherapeutic strategies, emerges as a promising avenue of investigation. In this part, we evaluated whether JWH133 enhances the chemotherapeutic efficacy of TMZ. Mice implanted with GL261-Luc cells were administered either JWH133 with or without concurrent TMZ therapy or a control vehicle. Tumor progression was assessed 14 days post-implantation through bioluminescence imaging using the IVIS. When comparing the TMZ treatment group to the control group, the study showed a significant decrease in GBM growth (Fig. 4A–B, P < 0.001). Interestingly, JWH133 with TMZ therapy significantly increased the anti-tumoral efficacy of TMZ alone (Fig. 4A–B, P < 0.01). The K-M survival analysis corroborated this inhibitory effect on tumor growth, showing that TMZ treatment alone was linked to a survival advantage over the control (Fig. 4C, P = 0.0031). Significantly, the co-treatment extended survival further than the TMZ treatment alone (Fig. 4C, P = 0.026). Collectively, these findings illuminate the potential of JWH133 to potentiate the therapeutic effectiveness of TMZ in combating GBM.