Flow cytometry efficiently detects ligand-induced G4 changes in HeLa cell
We have created a new FC-based approach to quantitatively measure G4 structure levels in cells (BG-flow). To validate this protocol, we measured if and how the FC signal is altered in cells incubated either with or without BG4 antibody (Fig. 1a). Cells were gated for size (forward scatter (FSC)) and granularity (side scatter (SSC)) as shown in Additional file 1: Fig.S1a. We detected that 81.4% of cells were positive for the BG4 signal. These analyses revealed a clear shift (based on mean fluorescence intensity (MFI)) of the BG4 signal (channel FL1) in comparison to the no BG4 controls, indicating that the antibody detected G4 structures in the cells. BG4 detection in IF as well as in FC was done with three antibodies. First, cells were incubated with BG4, then with anti-FLAG followed by a secondary antibody that harbored the fluorescent signal. Note, BG4 exhibits a FLAG epitope tag. In order to exclude false positive due to unspecific staining of these antibodies, we performed three additional controls: anti-FLAG + secondary antibody, BG4 + secondary, and only secondary antibody (Fig. 1a). Due to the nature of the BG4 antibody (a single-chain antibody), no isotype control is possible. These analyses already provided promising data that G4 structures can be detected by FC.
As a first benchmark for the capability of FC to detect G4s, we chose to compare its performance to the well-established IF detection of G4 structures in HeLa cells upon treatment with the commercially available G4-stabilizing ligand pyridostatin (PDS) [8, 11, 13, 14]. By IF, 10 μM PDS led to a 2.1-fold increase in G4 levels without affecting cell viability [11]. We repeated the published IF staining of HeLa cells that were incubated with either 1 μM or 10 μM PDS for 24 h. Similar to published data [11], 1 μM PDS caused no changes in G4 structure levels, whereas 10 μM resulted in a 1.75-fold increase in G4 structure abundance in comparison to untreated cells (Fig. 1b, Additional file 1: Fig. S1b). Note, in the IF, only the BG4 signal in the nucleus of the cells was quantified. The BG4 signal in the cytoplasm was very low and did not quantitatively alter G4 structure levels. To confirm the flexibility and robustness of the method, we addressed if FC can be used, similar to IF, to detect changes in G4 structure levels after stabilization with PDS. We treated cells with 1 μM and 10 μM of PDS (24 h) and analyzed G4 structure levels by FC. In agreement with the IF data (Additional file 1: Fig. S1a), FC analysis revealed no difference in BG4 signal after incubation with 1 μM of PDS but a clear shift in the histogram pattern after incubation with10 μM PDS (Fig. 1c, Additional file 1: Fig. S1c). Cells were gated for the size (FSC) and granularity (SSC) (Additional file 1: Fig.S1b). The quantification of the BG4 signal showed no fold changes compared to untreated cells for incubation with 1 μM PDS and a 1.8-fold increase for 10 μM PDS (Fig. 1d). These results were in agreement with results obtained by the established BG4 IF protocol, supporting the finding that FC analysis is a valid method to quantify G4 structures in fixed cells.
In order to exclude a bias due to PDS emission in FC, we expanded the analysis to another compound, PhenDC3. PhenDC3 is a bisquinolinium derivate that has a high affinity for G4 structures and stabilizes them in vitro and in vivo [15]. Two concentrations, 10 and 25 μM, were tested. Our analyses revealed an increase (based on MFI) of the BG4 signal (1.7-fold) in cell incubated with 25 μM PhenDC3 compared to untreated HeLa cells (Fig. 1e, Additional file 1: Fig. S1d). No changes were detected with 10 μM PhenDC3 (Fig. 1e, Additional file 1: Fig. S1d).
Methanol/acetic acid was used for the fixation of the cells both in IF and FC. Fixation with organic solvents, such as methanol/acetic acid, causes a severe loss of cell membrane integrity and cytoplasmic structures and the consequent loss of RNA G4 structures. In order to understand which is the contribution of cytoplasmatic RNA G4 structures to the overall G4 landscape, we performed the same analysis with paraformaldehyde (PFA) fixation. Both fixation methods have been used for IF [8, 11]. Untreated and PDS-treated cells (10 μM) were fixed with PFA (2% (v/v) in 1 × PBS for 15 min) and G4 structure levels were measured by FC (Fig. 1f, Additional file 1: Fig. S1e). Similar to methanol staining, we revealed a 1.6-fold increase of G4 structures after PDS incubation compared to untreated. Direct comparison showed a marginal increase (1.2-fold) after PFA fixation compared to methanol fixation (Fig. 1f, Additional file 1: Fig. S1e). This data indicated that a minor fraction of cytoplasmic G4 structures was lost due to the fixation with methanol/acetic acid. This change indicates that both fixation methods are working in BG-flow. Further, with methanol fixation, nuclear G4s are quantified, whereas with PFA both nuclear and cytoplasmic G4s are detectable. In the subsequent analysis, we performed methanol/acetic acid fixation, which allowed us to better compare the data to the IF where only the nuclear fraction was quantified (data not shown). Taken together, these results confirm the robustness of the BG-flow technique.
BG-flow can monitor G4 signal in THP-1 cells
To further validate and extend the working spectrum of BG-flow, we extended the analysis to THP-1 cells. THP-1 cells are human monocytes derived from a patient with acute monocytic leukemia. THP-1 are cells growing in suspension and often used as an in vitro cancer cell model [16], as well as a model to study the monocyte-macrophage differentiation process [17]. Similar to HeLa cells, incubation with BG4 revealed a clear shift (based on MFI) of the BG4 signal in comparison to the antibody-free control. This demonstrated that BG4 detected G4 structures in THP-1 cells (Fig. 2a, Additional file 2: Fig. S2a).
Because the effect of G4 structure stabilization by PDS has not been studied in THP-1 cells, yet, we first determined the cytotoxicity of PDS with MTT assay. We aimed to obtain a survival rate of at least 80% after 24 h to avoid measuring apoptosis/necrosis mechanisms, which likely involve G4 structures. We initially used 25 μM and 50 μM PDS (Additional file 2: Fig. S2b). THP-1 cells were incubated with PDS for 24 h and G4 structure levels were measured by FC and IF (see the “Methods” section). Similar to data from HeLa cells, the two approaches yielded very consistent and reproducible results. Using untreated cells as a reference, G4 structure level changed 1.5- and 1.6-folds (at 25 μM PDS) for IF and FC, respectively, and 1.7-fold for both at 50 μM PDS (Fig. 2b–d, Additional file 2: Fig.S2a, c). As seen with HeLa cells, only the nuclei were used for IF quantification because the signal from the cytoplasm was negligible (data not shown). These results clearly support the finding that BG-flow can be used to measure G4 structure levels also in suspension cells. We also demonstrated for the first time that G4 structures form in human monocytes and that PDS leads to an accumulation of G4 structures in these cells.
BG-flow can be used to determine cell cycle changes of G4 structures
We wanted to investigate, if BG-flow can be used to determine changes of G4 structure levels throughout the cell cycle in human cells as reported for IF [8]. We co-stained cells with BG4 and 4′,6-diamidino-2-phenylindole (DAPI). DAPI has been used before in FC to determine cell cycle phases [18]. MCF-7, an immortalized breast cancer cell line, was selected due to published data [8]. Similar to HeLa cells, the incubation with BG4 revealed a clear shift (based on MFI) of the BG4 signal in comparison to the antibody-free control. This indicated that BG4 detected G4 structures in MCF-7 cells (Fig. 3a, Additional file 3: Fig.S3a-b). By using the multidimensional cell cycle analysis package in FlowJo [19], we could discriminate each phase of the cell cycle based on the amount of DNA present in the cells indicated by the DAPI signal strength. Three different cell cycle phases were detectable: G0/G1, quiescent cells; G1/S, cells prone to enter in the replication phase; and S/G2, replicative cells (Fig. 2b). G4 structure levels were measured for each MCF-7 cell population and sorted by cell cycle phase. These data showed the highest levels of G4 structures in S/G2 phase (Fig. 3c): 1.5-fold more G4 structures than during G0/G1 phase (Fig. 2c). Here, we confirmed that in MCF-7 cells the cellular G4 landscape substantially varies throughout the cell cycle progression [8]. With minimal G4 structure levels during G0/G1 phase and a strong correlation between maximal G4 structure levels in the S/G2 phase [8, 20]. We next wanted to test if BG-flow can be used to determine G4 structure changes during the cell cycle in THP-1 cells. We co-stained THP-1 cells with BG4 and DAPI. G4 levels were then measured for each THP-1 cell population sorted by cell cycle phase. THP-1 cells showed higher G4 structure levels in both untreated and PDS-treated cells in the G2 phase (Fig. 3e). Note, upon PDS treatment, the increase of the BG4 signal was the same (~ 1.6-fold), regardless of the cell cycle phase (Fig. 3e). Upon PDS treatment, the G2 phase fraction strongly increased (from 15.9 to 54.4%) along with a comparable decrease of the G1 phase population from 51.4 to 12% (Fig. 3d). This data demonstrated that BG-flow can be used in double staining, to determine G4 structure levels during different cell cycle phases. In addition, we demonstrated that in THP-1 cells G4 structure levels peak in the G2 phase. It is not clear why G4 structure levels peak in the G2 phase in THP-1 cells. It could be that THP-1 cells, which are monocytic cells, require G4 structure formation during the G2 phase for a monocytic-specific function. In general, monocytes do not proliferate, are extremely sensitive to reactive oxygen species (ROS), and lack a functional base excision repair and DNA double-strand break repair via nonhomologous end joining [21]. In the G2 phase, the replicated DNA is not yet condensed and DNA is repaired mainly by homologous recombination [22]. One hypothesis is that G4 structures perform a function in the response to ROS in THP-1 cells. G4 structures could also support the differentiation process of monocytes into macrophages or dendritic cells. From the presented data, the function of G4 structures during the G2 phase cannot be explained, but the finding is of great interest and will be further studied.
BG-flow can be used to determine G4 structure levels in blood cells
Next, we tested whether BG-flow can measure G4 structure levels in peripheral blood mononuclear cells (PBMCs). PBMCs are nucleus-containing cells (predominantly lymphocytes and monocytes) isolated from human peripheral blood. We isolated PBMCs from buffy coats using a standard isolation protocol [23]. BG4-flow analysis was performed and BG4 signals quantified as before. Similar to HeLa cells (Fig. 1), BG4-incubated PBMCs revealed a significant shift in peak distribution in comparison to the antibody-free control (Fig. 4a, Additional file 4: Fig. S4a-b), but only 16% of the population showed a BG4 signal. This data indicated that G4 structures form only in a certain set of human blood cells. In order to support this finding, we performed IF in PBMC cells from a healthy donor and detected G4 structures in BG4-incubated PBMCs (Fig. 4b). Further analysis with specific biomarkers will be required to address which subpopulation forms G4 structures.
In most cancer cells, an increase of G4 structures was detected [7, 8, 10, 11, 13]. It is assumed that the amount of G4 structures correlates with cancer progression and the mutagenic burden of the cells [24]. Leukemia is triggered by the abnormal proliferation of blood cells. Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Several G4 ligands have been tested for the treatment of leukemia [25,26,27,28]. Interestingly, a bioinformatic analysis revealed that 70% of the genomic rearrangements in leukemia correlate with G4 motifs [29]. However, it is not known, if G4 structure formation is also enriched in AML cells. Therefore, we extended the previous analysis of blood cells and measured G4 levels in AML cells. We isolated PBMC from an AML patient with 99% of myeloid blasts in peripheral blood and performed BG-flow. PBMCs from a healthy donor served as a control. G4 structure levels in the AML cells were 2.17-fold lower than in healthy PBMC (Fig. 4c, d). Note, in this case, we already removed the background signal obtained in the negative control. This result needs further investigation to fully address its biological and possible clinical relevance. Note, AML PBMCs (here: 99% myeloid blasts) differ significantly from those of healthy individuals. It could therefore be that the observed decrease in G4 structures is not specific to recombination events in AML cells, but is based on different cell types. AML patients often have myeloid blasts, whereas healthy individuals have a heterogeneous composition of PBMCs (different lymphocyte subsets, monocytes, etc.). In summary, we could show that BG-flow is a fast and quantitative method to measure G4 structure levels in mixed cell populations of human blood cells.
PDS induces a G4 structure increase in mouse macrophages
We successfully applied flow cytometry-based analysis of G4 structures in HeLa and THP-1 cells as well as PBMCs. We extended our analysis also to other species and examined murine macrophages. Mouse macrophages derived from the bone marrow, spleen, and peritoneum are routinely used in the research of the innate and adaptive immunity [30]. For our analysis, we used an immortalized mouse macrophage cell line [31]. In detail, the macrophages were isolated from mouse tibiae and immortalized by using SV40 virus transformation. We determined G4 structure levels in these cells in comparison to the antibody-free control. A shift of the BG4-signal was observed in BG4-incubated cells as compared to the antibody-free control (Fig. 5a, Additional file 5: Fig. S5a). As with human cells, these results only account for the BG4 signal from the nuclei. We added analogous to our previous studies PDS to the cells to control the specificity of the signal for G4 structures. The working concentration of PDS was assessed by MTT assay. A 4-h treatment with 25 μM PDS led to more than 90% viable cells (Fig. S5b). To monitor the effect of PDS on G4 structure levels, the cells were incubated with 7 and 25 μM PDS for 4 h. First, we performed standard IF analysis, which revealed a 1.4-fold increase in BG4 signal upon 25 μM PDS and no change for the 7 μM PDS in comparison to untreated cells (Fig. 5b). The BG-flow approach led to a fold increase of ~ 1.7-fold, whereas 7 μM PDS led to a 0.95-fold change compared to the untreated control (Fig. 5c, d, Additional file 5: Fig.S5a, c). Taken together, these results confirmed the reproducibility of BG-flow also in mouse cells.