The G-quadruplex fluorescent probe 3,6-bis(1-methyl-2-vinyl-pyridinium) carbazole diiodide as a biosensor for human cancers

Using time-gated fluorescence lifetime imaging microscopy, significantly more signals from 3,6-bis(1-methyl-2-vinyl-pyridinium) carbazole diiodide (o-BMVC) foci, characterized by the longer fluorescent decay time of o-BMVC, were detected in six types of cancer cells than in three types of normal cells. Accumulating evidence suggested that the o-BMVC foci are mainly the G-quadruplex foci. The large contrast in the number of o-BMVC foci can be considered as a common signature to distinguish cancer cells from normal cells. Further study of tissue biopsy showed that the o-BMVC test provides a high accuracy for clinical detection of head and neck cancers.

Using time-gated fluorescence lifetime imaging microscopy, significantly more signals from 3, 6

-bis(1methyl-2-vinyl-pyridinium) carbazole diiodide (o-BMVC) foci, characterized by the longer fluorescent decay time of o-BMVC, were detected in six types of cancer cells than in three types of normal cells. Accumulating evidence suggested that the o-BMVC foci are mainly the G-quadruplex foci. The large contrast in the number of o-BMVC foci can be considered as a common signature to distinguish cancer cells from normal cells. Further study of tissue biopsy showed that the o-BMVC test provides a high accuracy for clinical detection of head and neck cancers.
Cancer remains as one of the leading causes of death in many countries. Since early diagnosis can improve the survival of cancer patient, the early detection of cancer has been an unmet and urgent medical need. Thus, finding a common target would be a great advantage to prevention, diagnosis, prognosis, and treatment in personalized medicine. Considering the great diversity in the root cause of different cancers, it is very challenging to comprehend the use of a common signature for all cancer types.
G-quadruplex (G4) structures formed by the stacking of G-quartets with Hoogsteen hydrogen bonding of four guanines have gained much attention as a possible target for cancer research 1,2 . The importance of G4 formation has been associated with genome instability, genetic diseases, and cancer progression [3][4][5][6] . Recently, a high-throughput sequencing-based method has identified 716,310 potential G4s in the human genome 7 . In addition, high density of G4s was found in the promoters, 5′UTR of transcribed genes, and splicing sites, and particularly in cancer-related genes 5,6 . We have previously used G4 fluorescent probe to demonstrate the existence of G4 structures in the metaphase chromosome 8 . Biffi et al. 9 have used a fluorescently labeled G4-specific antibody (BG4) to visualize the G4 foci in both cancer and normal cells. They found ~30% more in the number of G4 foci in HeLa cancer cells than in MRC-5 normal cells. Hansel-Hertsch et al. 6 found that immortalized keratinocytes (HaCaT) showed ~4-fold more G4 foci than do normal human epidermal keratinocytes (NHEK) using BG4 immunofluorescence microscopy. It appears that the difference in G4 foci between cancer cells and normal cells warrants further study.
Bioimaging is a powerful tool in visualizing cellular responses and monitoring target/probe interaction in cells 10,11 . Using imaging, fluorescent G4 ligands could provide a relatively simple, low-cost, and rapid approach to visualize G4s in cells. Among the limited available G4 fluorescent probes 12,13 , a 3,6-bis(1-methyl-2-v inylpyridinium) carbazole diiodide (o-BMVC) molecule has shown a higher binding affinity to G4 DNA than to duplex DNA by nearly two orders of magnitude 14 . The chemical structure of o-BMVC was shown in Fig. 1a. Of importance is that fluorescence lifetime imaging microscopy (FLIM) showed longer fluorescent decay times of o-BMVC upon interaction with most G4s formed by G-rich sequences in telomeres and some promoter oncogenes (≥2.4 ns) than with other structures such as linear duplexes and hairpin structures (~1.2 ns) 14

o-BMVC foci can act as a common biosensor of cancer cells.
We have used FLIM to measure the fluorescence decay time of o-BMVC upon interaction with 32 DNA sequences. All sequences were listed in Supplementary Table S1. The results showed that the decay times of o-BMVC are longer upon interaction with 20 G4s than with 10 sequences of duplex and single stranded DNA (Fig. 1b). Of interest was that one of the G-rich sequences, TBA-3G, could form a G-triplex with two G:G:G triad planes 15,16 , while one of the non-G-rich sequences, HD28, could form a triplex containing both T:A-T and C + :G-C triplets 17 . The FLIM results showed that the fluorescence decay time of o-BMVC upon interaction with TBA-3G was 3.1 ns, which was longer than ~1.5 ns upon interaction with HD28 but was similar to the decay time upon binding to G4s. Likewise to the G4 formation, a G-triplex is also a noncanonical DNA structure formed by the stacking of G-triplets. The G-triplet was made by Hoogsteen hydrogen bonds between guanine residues to stabilize G:G:G triad plane. Given that external stacking to the G-quartets is the major binding of o-BMVC to G4 14 , the 3.1 ns decay time measured from o-BMVC upon interaction with TBA-3G is also likely due to external stacking to the G-triplets. Given that TBA-3G was originally proposed as G4 folding intermediates of the TBA 18 , TBA-3G truncated from TBA was used to verify the presence of G-triplex 15,16 . Such G-triplex was also studied in human telomere 19,20 . Although limited G-triplexes have been documented, it is possible to have abundant G-rich sequences with 3 G-tracts to form G-triplex structures in human genome. Thus, more studies are necessary to establish the importance of G-triplex formation, such as the possible contribution from G-triplexes to the o-BMVC foci in distinguishing cancer cells from normal cells. Considering that the G4 structures are more stable than the G-triplex structures ( Supplementary Fig. S1) and the G4s are potential targets for cancer research, we therefore put our attention on the contribution from G4s to the o-BMVC foci in this work. o-BMVC foci are likely G4 foci. Rodriguez et al. 25 found that pyridostatin (PDS), a G4 ligand, not only induced DNA damage at sites enriched in G4 motifs but also affected the expression of genes that have G4-forming sequences within their promoters. They further visualized G4 foci in simian virus (SV40)-transformed MRC-5 human fibroblasts by optical imaging with Alexa Fluor 594 labeled PDS. Consistent with their finding, our quantitative analyses showed that marked increase in the number of o-BMVC foci was detected in PDS pretreated MRC-5 normal cells (Fig. 2a). Here the use of PDS without SV40 transformation could increase the number of G4 foci in MRC-5 normal cells. In addition, Biffi et al. 9 detected 2.9-fold more G4 foci in the PDS pretreated human U2OS cancer cells using the BG4 antibody. Consistent with their finding, we found that PDS increased the number of o-BMVC foci by ~2-fold in the nuclei of HeLa cancer cells (Fig. 2b). The agreement of these two  (Fig. 2c), which supported our hypothesis. Moreover, it is important to find that the number of o-BMVC foci induced by UV light showed appreciable decrease after the UV light-treated cells kept in the dark overnight (Fig. 2c). Further study by using BG4 antibody confirmed that DNA damage induced by UV-irradiation could generate more numbers of G4 foci in MRC-5 cells (Fig. 2d). This finding also supports that the o-BMVC foci are mainly the G4 foci.  (Fig. 3d). According to the number of o-BMVC foci results and the receiver operating characteristic (ROC) curve analysis, a threshold value of 7.5 was applied to differentiate malignant and benign specimens. Astonishingly, the ROC curve showed the area under curve (AUC) was 0.992 (Fig. 3e), indicating that this method provides a very high accuracy for detection of HNC cells from patients.

Validation of o-BMVC
In addition, we found that 12 of the 16 cases with high average number of o-BMVC foci had stage 4 disease, 1 had stage 3, 1 had stage 2, and the remaining 2 had stage 1 disease. The 2 patients that had the highest average number of o-BMVC foci were one with stage 4 nasopharyngeal cancer (double cancer, the sample was from the tongue) and one with stage 1 tongue cancer but recurred with stage 4 disease. It is probably that the average number of o-BMVC foci correlates with the tumor burden (disease stage), which deserves further studies.
HNC is the sixth most common cancer in the world 26 . Most HNC patients are found to have a locally advanced or metastatic tumor at the time of diagnosis. Despite the improvement of multidisciplinary treatment during the past decades, the survival of locally advanced HNC patients has not been improved 27 . On the other hand, for early stage HNC patients, the treatment outcomes are generally good 28 . It is thus important to develop better screening programs and novel diagnostic measures for early diagnosis of HNC to improve the survival of patients. In this study, we used HNC tumor tissues for validation of in vitro cell line findings is due to both tumor and control samples are easily accessible. Furthermore, this pilot study provides the possibility that o-BMVC test may be used for oral swabs in the minimally invasive diagnosis of oral cancers. Further large scale clinical trials are needed to verify this point.

Discussion
In this work, we used a G4 fluorescent probe of o-BMVC to stain fixed cells and found a large contrast in the number of o-BMVC foci between cancer cells and normal cells in time-gated FLIM images for the discrimination of human cancers. Our results are consistent with the previous study by using BG4 immunofluorescence microscopy. Biffi et al. 29 showed that the use of BG4 antibody could stain G4 DNA in patient-derived tissues using immunohistochemistry. They observed that there are a greater number of G4 foci in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Accordingly, it is likely that the large contrast in the number of G4 foci between cancer cells and normal cells may act as a common biomarker for human cancers. At present, we do not know exactly why there are more numbers of G4 foci in cancer cells than in normal cells. Given that loss of genomic integrity is a common hallmark of cancer 30,31 , we anticipated that the loss of chromatin integrity also plays an active role in the predominance of G4 foci in cancer cells because of either more G4 formation or easier for o-BMVC binding. Of importance is the gradual decrease of the induced o-BMVC foci after terminating UV-irradiation, suggesting that the G4 foci induced by DNA damage are different from the G4 foci detected in cancer cells. It is likely that the decrease of o-BMVC foci is due to the function of DNA repair. Considering impaired DNA repair is commonly occurred in many cancers 32 , this mechanism may play a major role in enriching the G4 formation in cancer cells.
Given that the etiology and clinical manifestation of different cancers are widely varied, direct evidence to confirm that the o-BMVC foci are the G4 foci is critical to validate the use of G4 foci as a common biomarker of cancer cells. To explore this possibility, one can determine the G4 sequences by gene sequencing and NMR and identify the surrounding proteins by mass analysis from isolated o-BMVC foci in cancer cells. Such study may shed new insights into G4 formation, identify mechanisms of G4 function, and unravel therapeutic targets of G4 related genes. Moreover, the determination of gene sequence can verify the potential contribution to the o-BMVC foci from the G-triplexes formed by the G-rich sequences with three G-tracts in human genome. At present, we are limited by the techniques to micro-dissect and to collect sufficient isolated o-BMVC foci. Nevertheless, the o-BMVC foci provide an opportunity for direct approach in verifying the use of G4 foci as a common cancer biomarker.
In summary, the significance of this study was to unequivocally demonstrate that many more o-BMVC foci are present in cancer cells than in normal cells. Although our tested cells are only small amounts of the vast cell lines, this finding lays the foundation for the development of o-BMVC foci as a common indicator of cancer cells. Consistent with the finding in the study of cell lines, the average numbers of o-BMVC foci are 28.3 from 50 head and neck patients and 2.2 from 20 healthy volunteers. Our findings suggest that the o-BMVC test is a novel assay for clinical screening of human cancers.
Quantitative measurement of o-BMVC foci is useful to establish a platform in monitoring cellular response under different conditions. For instance, DNA damage can induce the o-BMVC foci, while DNA repair may reduce the o-BMVC foci, implying that DNA repair plays a role in the large contrast of o-BMVC foci between cancer and normal cells. In addition, o-BMVC foci may serve as a convenient indicator to monitor carcinogenic transformation, which is essential to the development of early screening of cancer. Visualizing the change of o-BMVC foci may also lead to yield new insights into the underlying mechanisms of carcinogenesis and provide a new direction for early diagnosis and drug development in personalized medicine and biomedical research.

Methods
Chemical and sample preparation. The synthesis of o-BMVC can be found elsewhere 14 . All oligonucleotides purified by HPLC were purchased from Biobasic Inc. (Canada). Solutions of 10 mM Tris-HCl (pH 7.5) and 100 mM KCl mixed with each oligonucleotide were heated to 95 °C for 5 min, cooled slowly at 1 °C/min to room temperature and then were stored overnight at 4 °C before use. The concentration of each oligonucleotide was determined by UV absorption nanophotometry (Implen, Germany). Immunofluorescence. For immunofluorescence, MRC-5 cells were grown on glass coverslips without and with exposure to UV-irradiation and then were fixed with methanol/acetic acid (3:1) for 10 min. Fixed cells were permeabilized with 0.2% Triton-X100 and then were blocked with 2% Bovine serum albumin (BSA). After blocking, cells were incubated with a commercial BG4 antibody (Ab00174-1.1; absolute antibody, UK) for 1 h and then were incubated with anti-mouse Alexa Fluor 647-conjugated (A21235, Invitrogen, USA) secondary antibodies for 1 h. Samples were co-stained with 0.1 μM DAPI for 20 min and were visualized using a confocal microscope (Leica TCS SP8). Fluorescence excitation was carried out at 647 nm for Alexa Fluor 647 and 405 nm for DAPI. Quantitative analysis of o-BMVC foci. Since the fluorescent decay time is longer upon binding to G4s than other structures, the acquired FLIM results of o-BMVC in cells were presented in pseudocolor and separated into two channels: white (decay time ≥2.4 ns) and red (decay time <2.4 ns) to map the G4s. Here we used HeLa cancer fixed cells as an example (Fig. S3A, left). After excluding the non-signal pixels (intensity = 0), the gray-level histogram of the longer lifetime channel can be fit as the mixture of Gaussians (Fig. S3B). The optimal threshold was further determined by the Otsu algorithm to eliminate the weaker signals, which may be due to the loose binding of G4 DNA or the non-specific binding of small cell fragments, in the longer lifetime channel. The Otsu threshold method 21 was used to find an optimal threshold (T opt ) to separate two clusters or the mixture of Gaussians, with the following formula: where P(T) is the cumulative probability, m b (T) is the mean of the background, m f (T) is the mean of the foreground, σ b 2 (T) is the variance of the background and σ f 2 (T) is the variance of the foreground. After applying the Otsu threshold method, the weak signals can be eliminated, while the stronger signals (the red spots in Fig. S3A, right) can be preserved. The same imaging process and analysis were applied to the FLIM images of fixed cells and live cells. By using the algorithm for the image analysis, we can lower the possible counting errors in human eye detection and unambiguously quantify the number of foci in different cell lines.

ex vivo study of clinical cells.
For the study of normal oral samples, the buccal mucosa cells collected by Q-tips from healthy volunteers were fixed with 70% ethanol on microscope slides for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of head and neck cancer samples obtained during surgery, the cancer cells were fixed with 70% ethanol on microscope slides for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. After data collection, the receiver operating characteristic (ROC) curve was analyzed by web-based calculator 33 . Ethics Statement. This study was approved by the Institutional Review Board of National Taiwan University Hospital (NTUH REC No. 201304078RIND). Informed consent was obtained from all participants and/or their legal guardians. All methods were performed in accordance with the relevant guidelines and regulations.