Construction and evaluation of GFAP-IMLs
This study was to construct CNS neoplasms CTCs separation system based on GFAP antibody immunolipid magnetic liposomes and to verify it in brain tumors. GFAP-IMLs were prepared by reverse emulsification method by using GFAP-GHDC, DOPC and cholesterol. Fe3O4 nanoparticles were wrapped in liposomes, and the long chain alkyl part of amphiphilic GFAP-GHDC was inserted into the lipid bilayer membrane, and most of the GFAP antibodies were distributed on the surface of the liposomes. After CTCs separation of collected CSF or blood was performed by GFAP-IMLs, DAPI/EGFR-FITC combination immunofluorescence staining was used for identification and CTCs counting. Meanwhile, DNA extraction and gene testing of selected CTCs groups could be performed (Fig. 1).
The microstructure and physicochemical characterization of GFAP-IMLs were analyzed and compared with EpCAM-IMLs. Clear protein bands were observed in GFAP-GHDC and GFAP-IMLs in protein electrophoresis photo, indicating that the GFAP antibody was successfully inserted into the lipid bilayer of IMLs (Fig. 2A). The UV absorption spectrum of IMLs showed that the GFAP antibody, GFAP-GHDC and GFAP-IMLs had clear UV absorption peaks near 280 nm. However, with the modification of antibodies and the influence of nanospheres on UV absorption, the absorption peaks of the antibody derivatives and IMLs at 280 nm become weaker, wider and slightly shifted (Fig. 2B). The molecular configurations of GFAP antibody, GFAP-GHDC and GFAP-IMLs were studied by in situ electrochemical Raman spectroscopy. Raman spectra revealed the same characteristic peaks for all samples (Fig. 2C). The magnetic saturation curve showed that the prepared GFAP-IMLs had a high saturation magnetization, and no hysteresis was detected in the curves of Fe3O4 raw magnetic beads and GFAP-IMLs (Fig. 2D). The hysteresis curve was closed. The residual magnetic force and coercive force were zero within the allowed range of the instrument, suggesting good superparamagnetic properties. The maximum specific saturation magnetization of magnetofluid Fe3O4 was 51.3 emu/g and that of GFAP-IMLs was 30.9 emu/g, accounting for only 60.2% of the pure magnetofluid. The EpCAM-IMLs and EGFR-IMLs showed similar magnetic properties.
AFM revealed similar spherical shapes for these three IMLs, with relatively low distribution uniformity sizes (Fig. 2E-G). The particles were larger than 100 nm in diameter, and the surface of the beads was coarse. When the particle image was further enlarged (lower right corner), it was revealed that the shapes of these IMLs were irregular, and the magnetic bead surfaces had an antibody lipid membrane. The particle size and zeta potential of the three IMLs were not significantly different, as presented in the respective AFM images. A smaller particle size was more beneficial to the interaction between the beads and cells to improve the separation efficiency of CTCs.
As indicated in the cytotoxicity assay, the inhibition rate of the three IMLs constructed in this study on glioma cells was low at a concentration of < 100 µg/ml, and the cytotoxicity in glioma cells increased gradually as the concentration rose higher than 200 µg /ml. These results also suggested an equal inhibitory effect of the three IMLs on tumor cells, and the inhibitory ability was only related to the concentration of the IMLs added.
The cell inhibition rate of GFAP-IMLs was investigated by using U251 (Additional files: Fig. S1) and D425 (Additional files: Fig. S2) cells, respectively. The results showed that GFAP-IMLs had no significant inhibitory effect on the proliferation of the two cells for 0.5 h compared with untreated cells. With the extension of the action time, the cells were inhibited to a certain extent, but the survival rates were still more than 80% within 24 hours. Therefore, in the cell sorting experiment, we kept the interaction time between GFAP-IMLs and target cells within 0.5 hours.
The combination process between GFAP-ILs and U87 cells treated with fluorescent dye were observed by laser scanning confocal microscope (LSCM) (Fig. 3). The fluorescence intensity of U87 cells increased gradually over time, indicating an increase of the amount of GFAP-ILs labeled by FITC. The bright-field view demonstrated the reduction of GFAP-ILs outside the cells. A large number of GFAP-ILs were adhered on the cell surface at 10 minutes. The penetration of GFAP-ILs into the cells significantly increased after 20 minutes. Therefore, to separate the target cells, the incubation time of GFAP-IMLs could be limited in 30 minutes. Magnetic separation with a higher cell recovery rate was conducted in 20 ~ 30 minutes of incubation in the following experiment.
In order to further confirm the interaction between GFAP-IML and U87 cells, we selected GFP-labeled U87 cells and prepared R123-labeled GFAP-IML. Spontaneous signals of GFP in U87-GFP cells were observed from 0 to 30 minutes through immunofluorescence. At 5 minutes of incubation time, R123 could not be observed. However, with the extension of incubation time, the red fluorescence became visible and gradually enhanced. At 15 minutes, the R123 fluorescence was not strong enough, but the cell contour could be seen, indicating that the R123-GFAP-IMLs began to enter the cell interior. The entire cell contour was clearly visible at 25–30 minutes, suggesting that the R123-GFAP-IMLs had accumulated inside the U87 cells (Additional files: Fig. S3).
The results showed that three IMLs were able to capture U87 cells suspended in PBS under different concentration gradients. At the same antibody content on IMLs with the same magnetic quality, the average efficiency of GFAP-IMLs, EpCAM-IMLs, EGFR-IMLs and IMLs in capturing U87 was 87.9%, 63.8%, 49.4% and 30.8%, respectively. The capture efficiency of GFAP-IMLs in PBS was higher than those of EGFR-IMLs and EpCAM-IMLs (Fig. 4A). Children medulloblastoma D425 was also selected to evaluate the cell separation efficiency for three IMLs. The results showed that the average separation efficiency of GFAP-IMLs, EpCAM-IMLs, EGFR-IMLs and IMLs was 92.4%, 25.8%, 91.2% and 17%, respectively (Additional files: Fig. S4). It also indicated that D425 was highly expressed for GFAP and EGFR. There was no significant difference in the separation efficiency between GFAP-IMLs and EGFR-IMLs, which indicated that cytoplasmic protein GFAP can be selected as medulloblastoma cell separation target.
U87 cells were subcutaneously injected into 4-weeks-old nude mice. Three kinds of IMLs were used to mimic the capture of CTCs in the blood from nude mice after 3, 15, 30 and 40 days of subcutaneous injection. All IMLs could capture CTCs in the blood, and the number of CTCs captured by GFAP-IMLs was significantly higher than those captured by EGFR-IMLs and EpCAM-IMLs (Fig. 4B). Fluorescence images also showed that CTCs captured by GFAP-IMLs from the blood and tumor tissues of nude mice were similar in cell morphology to U87-GFP cells with spontaneous green fluorescence (Fig. 4C). The numbers of CTCs captured were positively correlated with the sizes of inoculated tumors, which were 0 mm3, 50 mm3, 100 mm3, and 200 mm3 in nude mice. In addition, when the tumor volume exceeded 50 mm3, the numbers of CTCs isolated by GFAP-IMLs were significantly higher than those isolated by the other two IMLs (p < 0.05). Through in vivo imaging of nude mice, it was confirmed that the tumor was composed of U87 cells with GFP green fluorescence (Fig. 4D).
Preliminary clinical application of IMLs in brain tumors
Thirty-two children (3 abnormal data points, which were much higher than the average, were deleted) with brain tumors were recruited in this study (Additional files: Table S1). GFAP-IMLs were applied to capture CTCs in both peripheral blood and CSF from these patients. EpCAM and EGFR IMLs were used as the control group. As a verification approach, captured CTCs were labeled by anti-EGFR antibody conjugated FITC and anti-CD45 antibody conjugated by PE. The patients’ CTCs images with GFAP high expression isolated from blood and CSF are shown (Fig. 5). All cells are 15 ~ 20 µm in size, with strong blue (DAPI) and green (EGFR-FITC) fluorescence. There are also obvious round and oval cell morphology under the white light, and the cell surface is covered with light brown magnetic particles (GFAP-IML nanoparticles).
The numbers of captured cells in peripheral blood and CSF from each sample, together with those in the control group, were calculated and summarized in the scattergram and heat map. The number of CTCs from embryonal tumors was lower overall than any other types of tumor (Fig. 6A). For other brain tumors, it was interesting to find that in pilocytic astrocytoma (PA) cases with KIAA1549-BRAF fusions, more CTCs were recruited than those in wild-type cases. Additionally, more CTCs could be captured in CSF samples than in peripheral blood samples from PA patients (Fig. 6B & C). These results indicated that the CTCs separation system might be a minimally invasive procedure for diagnosing children with images revealing suspected PA cases. Furthermore, this system may provide a novel method for posttreatment assessment of efficacy. In medulloblastoma, group 4 cases tended to have more CTCs than non-group 4 cases in both CSF and peripheral blood samples (Fig. 6D & E). This finding indicated that group 4 medulloblastoma had a likely higher frequency of metastatic. In addition, one patient exhibiting cerebellar glioblastoma with CSF dissemination also showed a higher CTCs number (Additional files: Fig. S5).
Of course, there was no significant difference in the CTC cells sorted by different immunomagnetic spheres in the blood (Fig. 7A&B). In CSF, the number of CTCs sorted by the GFAP immunomagnetic sphere was significantly higher than that of the EpCAM and EGFR magnetic spheres (P < 0.01). To our surprise, the number of CTCs sorted by GFAP magnetic spheres was not significantly associated with tumor stage, either in cerebrospinal fluid or in blood (Fig. 7C). The number of CTCs sorted by GFAP magnetic spheres was related to the age of the children (Fig. 7D). Children less than 3 years old had more CTCs in their CSF than those in other age groups (P < 0.01). Survival data was available for 60 brain tumor patients (Additional files: Table S2), and the median survival analysis was performed for three number cut-off of ≤ 10, 10–100 and ≥ 100 CTCs per 7.5 mL of CSF. The median survival time was 27 months when the number of CTCs was ≤ 10, and the median survival time was 15 months when the number of CTCs was within the range of 10–100. But when the CTCs number was above 100, the median survival was only 9.5 months. There were significant differences among the three CTCs number interval (P < 0.001) (Fig. 7E). It can be deduced that there is a significant correlation between the number of CTCs in CSF and the patient’s survival time. The higher the CTCs number in CSF is, the more possibly the patient will suffer from poor prognosis.
To strengthen evidences in supporting the origination of isolated cells, distinct genetic alterations were tested in CTCs according to the genetic characteristics in original tumors. We found that the H3F3A gene showed K27M mutations in CTCs (captured from CSF) and in tumor tissue samples from DIPG patients (Fig. 8). What’s more, CTCs from a patient diagnosed with medulloblastoma were revealed with over-expressed NPR3 by RT-PCR, which was consistent with immunohistochemistry staining of NPR3 protein in tumor tissue (Fig. 8C). What’s more, two same gene mutations of KMT2A and TMPRSS2 were checked out in GFAP separated CTCs and tumor tissue. Cells isolated from the GFAP-IMLs were further verified by whole exon sequencing and compared with the tissue (Additional files: Table S3). Blood sorting CTC detected 5 gene mutations and tissue detected 4 gene mutations, all of which were KMT2A and TMPRSS2 mutations, which again demonstrated that the cells sorted by self-made GFAP-IMLs were consistent with the tissues in some genes. In addition, the blood samples detected a code-shifting mutation in exon 20 of CIC gene, with a richness of 9.62%, which may lead to truncation mutation of the protein, thus affecting the protein function.