Cross-Platform Cancer Cell Identification Using Telomerase-Specific

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Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids Zhengjie Liu, Jun Zhao, Ruilong Zhang, Guangmei Han, Cheng Zhang, Bianhua Liu, Zhongping Zhang, Ming-Yong Han, and Xiaohu Gao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00743 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids Zhengjie Liu,†,‡,# Jun Zhao,†, # Ruilong Zhang,§ Guangmei Han,†,‡ Cheng Zhang,† Bianhua Liu,† Zhongping Zhang,*,†,§Ming-Yong Han,†,∆ and Xiaohu Gao*,¶ †

CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines, Chinese Academy of

Sciences, Hefei, Anhui 230031, China. ‡

Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026,

China. §

School of Chemistry and Chemical Engineering, Anhui University, Hefei, Anhui 230601, China.

∆

Institute of Materials Research and Engineering, A-STAR, 3 Research Link, Singapore 117602.



Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT: Distinguishing tumor cells from normal cells holds the key to precision diagnosis and effective intervention of cancers. The fundamental difficulties, however, are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers. Here, we report a concept of tumor cell detection, bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going towards the hallmark of cancer, uncontrollable growth. Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms,

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ranging from solution-based assay, to single cell imaging and in vivo solid tumor imaging (unlike PCR that is restricted to cell lysates). We envision the SNAB technology will impact cancer diagnosis, therapeutic response assessment, and image-guided surgery. KEYWORDS: telomerase, spherical nucleic acids, cancer, molecular beacons, surgery navigation, imaging, detection Uncontrollable growth (immortalization) is the hallmark of cancer, but identification of tumor cells versus normal ones has been a long-standing problem despite its significant impact on cancer diagnosis, treatment and prognosis. For example, specific imaging of solid tumor in vivo allows early cancer detection and tumor margin identification during surgery, whereas enumeration of circulating tumor cells (CTCs) reflects the efficacy of oncological interventions. A variety of enabling molecular profiling technologies such as single cell sequencing,1 molecular imaging,2 mass spectrometry,3 and immunohistochemistry (IHC),4 have been invented to help address this technical challenge. However, due to the heterogeneity of tumor cells and the lack of truly specific cancer biomarkers, none of these technologies is capable of identifying all tumor types with high accuracy, low cost, high throughput, and compatibility across the molecular, cellular, and whole-body scales. We set out to develop a technology that can bypass the specific genotypic and phenotypic features of various tumor cell types, and directly go to the hallmark of cancer, uncontrollable growth. Cell uncontrollable growth requires elevated telomerase activity. Telomeres at the end of chromosomes are a special structure protecting genome integrity from degradation, rearrangements and end-end fusion.5-8 Telomerase is inactive in normal somatic cells, but highly activated in most tumor cells to maintain their immortal phenotype and indefinite proliferation.9-14 Similarly, stem and progenitor cells also express detectable levels of telomerase for self-renewal, offering further evidence that telomerase is a necessary regulator of tumor growth.15-17 As a result, telomerase activity has become an increasingly important biomarker for tumor occurrence and progression. Indeed, the ACS Paragon Plus Environment

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potential of using telomerase activity as a cancer detection mechanism has been realized by a number of scientists. For example, Kim,10 Plaxco18 and Weizmann19 utilized PCR to detect telomerase activities in cell and tissue lysates; whereas Ju and coworkers20,21 attempted imaging telomerase activity in live cells, which would allow cancer diagnosis in vitro and in vivo. Unfortunately, the success has been limited due to the probe’s inefficient cell entry (lacking the SNA architecture), difficulty loading primer into telomerase (primer is double-stranded), and incomplete fluorescence activation (fluorophore still attached to quencher AuNPs after recognition of telomerase). Here, we show a simple yet effective combination of spherical nucleic acids (SNAs) and exquisitely engineered molecular beacons (spherical nucleic acid-beacon, dubbed SNAB) simultaneously achieves all the desired requirements for intracellular imaging of telomerase activity in live cells and lab animals. SNAs are nanoparticles coated with high-density DNA strands, often in a crew-cut organization.22,23 These coated nanoparticles have a number of fascinating attributes, and in particular the ability to enter virtually all types of cells without the need of transfection reagents.24-27 Using 13 nm Au nanoparticles (AuNPs), Mirkin and coworkers demonstrated that the cell uptake of AuNPs increases sharply with increasing DNA strand density and plateaus around 60 strands/particle.28 Taking advantage of this cell-entry behavior, we immobilized telomerase-targeting molecular beacons onto AuNPs surface to prepare a series of SNAs, SNAB probes. As shown in Figure 1A, the molecular beacon is consisted of a long telomerase primer (TP)-carrying strand and a short fluorophore-modified DNA strand (FL-strand) that are pre-hybridized before immobilizing onto AuNPs surface via the robust Au-thiol linkage. The TP-carrying strand has three functional domains: a TP sequence (18bp, underlined) for specific recognition of the telomerase catalytic core,29,30 a (CCCTAA)3 sequence (18bp, blue) complementary to the telomeric repeat,20,31 and an additional sequence of TTTTTGCAGC in which GCAGC is partially complementary to the FL-strand and T5 is a spacer to help reduce steric hindrance. The FL-strand (3′-CGTCGGGGATTG-5′) contains a fluorophore at its 3′ end, which is initially quenched due to close proximity to the AuNPs. In the presence of telomerase, the TP-carrying ACS Paragon Plus Environment

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strand is recognized by the catalytic core of telomerase, and elongated with telomeric repeat TTAGGG, resulting in formation of a more thermodynamically stable DNA hairpin structure. The FL-strand is subsequently displaced into the solution, restoring its fluorescence for detection (Figure 1B). RESULTS Probe Engineering and Characterization. Because the DNA density on SNAs is critical to cell entry, we first quantified the number of DNA beacons per AuNP by displacing them with mercaptoethanol.32 The concentrations of the DNA strands and AuNPs can be determined by their characteristic fluorescence emission and UV-vis extinction profiles, respectively (Figures S1, S2). Our calculation indicated that on average the 13 nm AuNPs each had approximately 70 duplex DNA chains on the surface, higher than the DNA density threshold for efficient cell entry previously determined by Mirkin and coworkers.28 Before and after DNA coating, zeta potentials of the AuNPs changed from 6.1 to -19.1 mV (Figure S2D). To understand the length of telomeric repeats needed for displacing the FL-strand, pre-synthesized (TTAGGG)n repeats (n = 1-5) was incubated with the SNAB probe. Figure S3 showed that a minimum of 3 repeats was needed for FL-strand release and significant fluorescence enhancement. To characterize the performance of the SNAB probe, we first measured its fluorescence switching behavior in buffers spiked with telomerase of known concentrations (Figure 1C). The fluorescent signal of SNAB probe initially increased linearly with increasing telomerase concentrations and eventually leveled off. At the telomerase concentration of 1 IU/L, the SNAB probe fluorescence was enhanced by 3.3 folds, whereas at the concentration of 20 IU/L the enhancement factor reached 17. More importantly, the “turn-on” fluorescence was so bright that it could be visually observed when the samples were illuminated with a 633 nm laser (Figure 1C, inset), offering potential for quick and lowcost qualitative detection.

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Similar experiments were also repeated using HeLa cell lysates, in which heat-treated HeLa cells were used as the control (telomerase deactivated, Figure S4). Remarkably, as few as 100 HeLa cells could be reliable detected based on the enhanced fluorescence (Figure 1D, P = 0.006) (Figure S5). To rule out the possibility that the ‘turn-on’ fluorescence was due to nucleases or other environmental factors (e.g., buffer makeup and pH), the SNAB probes were placed in solutions with various nucleases, buffers, and pH values. As shown in Figures S6, S7A, the SNAB probes were insensitive to pH fluctuations (pH 5-10), cell culture media, human plasma, and nucleases, free of false-positive sensor responses. The robust performance is perhaps not surprising, since AuNP is known for its ability to help protect immobilized DNA strands.33 This protection effect was also observed in our experiments when the SNAB probes were compared with free oligonucleotide probes without the AuNP (Figure S7B). It is worth mentioning that the oligonucleotide sequences used in the above studies have been systematically optimized for sensitively and specifically sensing telomerase activity. In particular, the length of the displacement FL-strand is a critical factor determining the stability, specificity, and fluorescence enhancement factor of the probe. A number of FL-strands of different lengths (8, 11, 12, 13 and 18 bp) were made and hybridized onto the TP-carrying strand, and the melting temperatures of the duplexes were characterized with fluorescent spectroscopy (Figure 2A, B). When the length of FLstrand is shorter than 11 bp, the melting temperature is below 37 °C, unsuitable for biosensing and imaging uses under the physiological condition (dehybridization resulting in false positivity, inset of Figure 2A). When the FL-strand is 12-bp long, its melting temperature reaches 40.5 °C, slightly above the physiological temperature. We show that this FL-strand length is sufficient in maintaining the stability of SNAB probe in biological environments. Indeed, when the FL-strand probes (8, 11, 12, 13 and 18 bp) were exposed to HeLa cell lysates, they exhibited fluorescence enhancements of 1.1, 6.1, 19.2, 16.7 and 1.4 folds, respectively (Figure 2B, inset). As the FL-strand became longer, the probe was sufficiently stable in the biological environment, but the sensing response diminished and was ACS Paragon Plus Environment

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essentially eliminated at 18 bp. This inefficient strand displacement can be explained by calculating the change of Gibbs free energy before and after strand displacement (Table S1). Based on this study, the 12 bp FL-strand was used for all the following studies. The scientific mechanisms underlying the outstanding SNAB sensor response are from two ultrafast biological processes, telomerase-catalyzed synthesis of telomeric repeats and strand-specific displacement.34,35 Confirmation of the Telomerase Sensing Mechanism. To verify the working mechanism of the SNAB probe, the TP-carrying strands were treated with telomerase, or lysates from HeLa cells and normal cell MRC-5, respectively. The products were amplified using PCR and subsequently analyzed with gel electrophoresis (Figure 2C). In the cases of telomerase and HeLa cell lysates, a series of electrophoretic bands appeared due to elongation of the TP-carrying strand, while the original TPcarrying strand disappeared, suggesting that different numbers of telomeric repeats were added onto the TP end. In contrast, the elongated TP-carrying strands were not detected when normal cell lysate was used. These results confirmed specific catalytic elongation of TP by active telomerase. Next, we also examined the FL-strand release using gel electrophoresis. In this experiment, fluorescein (green fluorescence) was linked to the FL-strand for easy observation. The duplex DNA was again incubated with telomerase, HeLa cell lysate, and MRC-5 cell lysate in the presence of dNTPs (Figure 2D). The products were directly analyzed by gel electrophoresis without PCR amplification. For telomerase and HeLa cell lysate, a strong band for the 12-bp FL-strand was detected on the gel; while the original band of the duplex DNA became very faint, indicating disassociation of the FL-strand as illustrated in Figure 1A. For normal cell lysate, however, the duplex DNA band remained unchanged and the FL-strand band was not seen. Taken together, these results confirmed the proposed mechanism of telomerase detection based on strand displacement. Imaging Telomerase Activity in Live Cells. Due to the cell entry capability of the SNAB probe, it allowed telomerase detection beyond homogenized samples (unlike PCR). Upon incubation with the SNAB probe, confocal microscopy revealed that the intracellular fluorescence of HeLa cells could be ACS Paragon Plus Environment

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directly monitored without removing the free probe in cell culture media because the free probe remained in the fluorescence turn-off state in the absence of telomerase, potentially enabling real-time imaging. As shown in Figure 3A, the cell fluorescence gradually increased with increasing probe concentration and plateaued around 6 nM probe. Similarly, when the probe concentration was fixed at 6 nM, the intracellular fluorescence also increased with longer incubation time and plateaued around 90 min (Figure 3B). The patterns of fluorescence inside cells were generally homogeneous, indicating probe escape from the endosome. Although the exact mechanism of endosomal escape and the quantity (percentage) of SNAB probes released to the cytosol are unknown, similar results have been previously observed for SNAs in siRNA and ODN deliveries.36-40 To confirm that the fluorescence enhancement observed was indeed induced by active telomerase in tumor cells, two control experiments were conducted. First, an alternative TP-carrying strand was designed by changing two bases (TT at the TP end changed to AA). As shown in Figure S8, no turn-on fluorescence was observed in HeLa cells, proving the probe sequence specificity. Second, HeLa cells were treated with a telomerase inhibitor, 3’-azido-3’-deoxythymidine (AZT), before incubation with the SNAB probe (Figure 3C). The turn-on fluorescence reduced in a dose-dependent fashion according to AZT concentration (completely disappeared at 20 µM AZT), confirming target specificity. To test the central hypothesis that tumor cells can be distinguished from normal cells by their telomerase activity that is directly related to cell uncontrollable growth, thus bypassing the heterogeneity problem of tumor cells, the telomerase SNAB probe was applied to 10 human tumor cells lines originally obtained from cervix, kidney, bladder, pancreases, mouth, breast, stomach, ovary, liver and lung, and 5 common somatic control cells (MRC-5, HFL1, QSG-7701, MCF 10A and HFF-1). Qualitative fluorescence images in Figure 4 and Figures S9-S11 showed that the tumor cells all responded to SNAB probe treatment and exhibited various degrees of fluorescence enhancement, whereas the five control cell lines did not yield detectable signal increase. This result was also confirmed with quantitative flow cytometry data (Figure 4C), potentially offering a mechanism for ACS Paragon Plus Environment

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quantitative characterization of the degree of tumor cell malignance since telomerase activity is tightly tied to the malignancy of tumor cells.41-44 Lastly, we evaluated the toxicity effect of the SNAB probe using the standard MTT assay before moving onto in vivo experiments. As shown in Figure S12, when HeLa cells were treated with the SNAB probes (6 nM, the common concentration used in majority of the current experiments) for various time durations (up to 24 h), cell viability was largely not affected. This can be attributed to the key components of the SNAB probe that are either biological (e.g., DNA) or bio-inert (e.g., AuNP). Similarly, when the concentration range of SNAB was expanded, cytotoxicity was still negligible, even at 30 nM (5x of the concentration used in the imaging studies). In Vivo Visualization of Solid Tumors. Imaging is an important part of cancer diagnosis and treatment. Visualizing tumors can help detect, stage cancer, predict prognosis, and stratify treatment plans. In recent years, advanced molecular probes have also captivated surgeons for intraoperative imaging, aiming for complete excision of tumor mass with minimum damage to healthy tissues and their biological functions. The key to achieving this goal is probes capable of distinguishing between tumor cells and normal cells during surgery. Here, we explored the possibility of translating the in vitro assays discussed above into an in vivo solid tumor imaging tool. Xenograft cervical tumor models in athymic nude mice were established by planting HeLa cells into the subcutaneous armpit area of mice. After the tumors reached size of 300 mm3, the SNAB probe was injected into the tumor (red circle) as well as the subcutaneously under the other arm (black circle). As shown in Figure 5A, red fluorescence signal started to appear 40 min post injection only on the tumor side. The fluorescence signal gradually increased over time and covered the entire tumor mass without spilling into the surrounding areas. In contrast, fluorescence remained undetected on the other side where tumor mass is absent. Localized probe activation in tumor was confirmed by exfoliating mouse skin near the tumor site (Figure S13). Similar results were also achieved on mice bearing lung and liver tumors (Figure S14).

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To confirm the in vivo imaging results, frozen tumor sections were obtained and examined with fluorescence microscopy. Remarkably, high resolution micrographs showed that only tumor cells in the exercised tissue are fluorescent, but not the intercellular space (Figure 5B). The results indicate that the SNAB probes are specific to intracellular telomerase activity, and potentially can provide guidance for surgery. DISCUSSION Distinguishing tumor cells from normal cells with high specificity has the potential to transform cancer diagnosis and treatment. However, truly specific and universal biomarkers do not exist. The heterogeneity problem of tumor cells adds another level of difficulty since a biomarker or a panel of biomarkers is only capable of spotting one type of tumor cells. To help address this issue, we proposed to bypass the detailed genotypic and phenotypical typing of tumor cells, and directly target uncontrollable cell growth, the hallmark of cancer. Intracellular telomerase activity was thus identified because it is a critical step in uncontrollable cell growth and is observed in 85-90% tumor cells.9,45 Targeting intracellular events in live cells, however, represents a fundamental challenge, because i) nanosensors based on nanoparticles or biomolecules generally cannot cross the cell membrane without transfection agents, and ii) constitutively-active probes cannot be washed away resulting false positive detection. These technical challenges were solved using the exquisitely designed SNAB probe which combines two powerful technologies, SNA and molecular beacon. SNA is known for their ability of carrying highly anionic biomacromolecules cross cell membranes. Detailed fluorescence and electron microscopy work has revealed that the rapid cellular uptake and intracellular transport of SNA stem from its binding to class A scavenger receptors and endocytosis via a lipid-raft–dependent, caveolaemediated pathway,46 whereas the molecular beacon remains non-fluorescent in the absence of target molecules. In addition, the combination of SNA with molecular beacon also helps stabilize the ACS Paragon Plus Environment

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molecular beacon (low background) because the steric hindrance and local high salt concentration caused by the high-density DNA on AuNP surface reduce nuclease activities.47 The molecular beacon sequences were also optimized through multiple rounds of iterations. In the final design, the distal end of the TP-carrying strand from AuNP remains single after hybridizing with the FL-strand. This single stranded tip facilitates substrate loading to inner catalytic core of telomerase. Besides the systematic study of the optimal number of telomeric repeats discussed in the results, a seemingly minor factor is the release of the FL-strand in the presence of telomerase rather than simply opening the hairpin ring in a typical molecular beacon (still keeping the extended molecular beacon on AuNP surface). However, this feature enhances the signal strength by avoiding partial quenching due to proximity to AuNP and self-quenching due to crowded fluorophores on nanoparticle surface. The robust performance and the flexibility of the SNAB probe enable highly specific detection of telomerase activity applicable across a number of detection platforms ranging from solution phase sensing, to intracellular imaging of single cells and intratumor imaging in live animals. Under in vitro conditions, an interesting observation is that although all tumor cell lines show significantly positive telomerase activity, their levels are different. This may potentially become a mechanism to quantitatively characterize the degree of malignancy or aggressiveness of tumor cells and compare them on the same chart. This systematic comparison is beyond the scope of the current work, but deserves future investigation which will involve culturing of a large number of cell lines (perhaps 100s1000s, beyond the 10 shown here) and characterizing their growth rates both in vitro and in vivo. Furthermore, the flexibility of the SNAB probe allows telomerase activity to be imaged in live animals, unlike PCR. In a simple model study, we demonstrated specific tumor cell imaging through intratumor injection in live animals. High resolution microscopy study of exercised tissues revealed that the telomerase activity was highly confined inside tumor cells. This localization behavior of SNAB probes allows tumor cell identification, which may potentially impact intraoperative imaging, leading to accurate determination of tumor margins. ACS Paragon Plus Environment

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CONCLUSION In conclusion, we have developed a universal platform distinguishing between tumor cells and normal cells based on the concept of focusing on biomarkers directly involved in uncontrollable cell growth, the hallmark of cancer. It is achieved by designing a series of SNAB probes intelligently combining two innovative technologies, SNA and molecular beacons, thus enabling analysis of intracellular targets. The SNAB technology outperforms the gold standard method of telomerase activity measurement using PCR in terms of flexibility because it is applicable to both single cells and solid tumors in vivo (beyond cell lysates). This intracellular imaging technology and the concept of imaging cancer based on uncontrollable cell growth are expected to impact medical diagnostics, therapeutic response assessment, and image-guided surgery. MATERIALS AND METHODS Animal use protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Hefei Physical Science (Chinese Academy of Science). Materials. All oligonucleotides used in this paper were synthesized by Sangon Biotech (China). The thermodynamic parameters and secondary structures of oligonucleotides were calculated using a bioinformatics software, Primer Premier 5.0 (PREMIER Biosoft International). Chloroauric acid (HAuCl4·4H2O, 99.9%), trisodium citrate (Na3C6H5O7·2H2O, 99.8%) and dimethyl sulphoxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 4',6-diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were supplied from Beyotime Biotech. Co. Ltd. (China). 3′-Azido-3′-deoxythymidine (AZT) was obtained from Sigma-Aldrich Inc. (USA). Deoxynucleotide solution mixture (dNTPs), RNase inhibitor, DEPC-treated water, Deoxyribonuclease I (DNase I) and Exonuclease III (Exo III) were bought from TaKaRa Bio Inc. (Dalian, China; DEPC = diethylpyrocarbonate). A TRAPEZE telomerase detection kit (S7700) ACS Paragon Plus Environment

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was purchased from Chemicon International, Inc. (Billerica, MA). Hybridization buffer (HB, pH 7.4) containing 10 mM Tris-HCl, 1mM EDTA, 50 mM NaCl and 10 mM MgCl2, phosphate buffer saline (PBS, pH 7.4) containing 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4 and 1.41 mM KH2PO4, and 5× TBE buffer (containing 445 mmol/L Tris-Boric Acid, 10 mmol/L EDTA) were received from Sangon Biotechnology Co., Ltd (China). These reagents were used without further purification unless specifically mentioned. Aqueous solutions were prepared using DI water (≥18 MΩ, Milli-Q, Millipore). Probe Preparation. 13 nm Au nanoparticles (AuNPs) were synthesized by reduction of HAuCl4 with sodium citrate according to reported methods32 and stored at 4 °C. TP-carrying strand, 5′-HSTTTTTGCAGCCCCTAACCCTAACCCTAAAATCCGTCGAGCAGAGTT-3′ was incubated with the FL-strand Cy5-3′-CGTCGGGGATTG-5′ with equal molar concentration in hybridization buffer (HB, pH 7.4). The mixture was briefly (5 min) heated to 90 °C under shaking, and slowly cooled to room temperature. The duplex DNA was treated with tris(2-carboxyethyl) phosphine hydrochloride to disassociate any potential dimers, and mixed with the AuNPs solution at a molar ratio of 300 under shaking for 16 hours. Phosphate buffer (0.1 M, pH = 7.4) and sodium chloride solution (2.0 M) were sequentially added into the above mixture to achieve the final concentrations of 0.01 M phosphate and 0.3 M sodium chloride. The probes were isolated after washing with PBS buffer three times. Cell Cultures. HeLa (human cervical carcinoma cells), 293T (human embryonic kidney cells), A549 (human lung carcinoma cells), MCF-7 (human breast carcinoma cells), KB (human oral carcinoma cells), NIH:OVCAR-3 (human ovarian carcinoma cells), N87 (human gastric carcinoma cells), 5637 (human bladder carcinoma cells), BXPC-3 (human pancreatic carcinoma cells), HFF-1 (human skin fibroblasts), and HFL1 (human embryonic lung fibroblast) were supplied by Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Science, China). MCF 10A (human breast cells) and QSG-7701 (human liver cells) were purchased from Shanghai Bioleaf Biotech Co. Ltd. Hep G2 (human liver carcinoma cells) was obtained from Center of Medical Physics and Technology, Hefei ACS Paragon Plus Environment

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Institutes of Physical Science (Chinese Academy of Science). HeLa, 293T, A549, MCF-7, HFL1, 5637, BxPC-3, N87, NIH:OVCAR-3 and MRC-5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillinstreptomycin. Hep G2, KB, MCF 10A and QSG-7701 cells were cultured in 1640 medium with 10% FBS and 1% penicillin-streptomycin. HFF-1 cells were cultured in DMEM medium supplemented with 15% FBS, 1% penicillin-streptomycin and 1% sodium pyruvate. The cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2) and were kept in logarithmic growth phase by routine passages every 2-3 days. Prior to the use of cells, the densities of cells were determined using a hemocytometer. Preparation of Cell Lysates. Cells were lysated by the NP-40 method according to well-established protocols.20 Briefly, cells were trypsinized from culture dishes, washed twice with PBS (pH 7.4), and resuspended in 1 mL of cooled lysis buffer (10 mM Tris-HCl, 1% NP-40, 0.25 mM sodium deoxycholate, 10% glycerol, 150 mM NaCl, 0.1 mM AEBSF, pH 8.0) at a concentration of 1.0 × 107 cells/mL. The cells were kept at 4 °C for 30 min under shaking, and the mixture was centrifuged at 11,000 rpm for 20 min at 4 °C to remove any cell debris. The lysates were flash frozen in liquid nitrogen and stored in -80 °C for further uses. Cytotoxicity Evaluation. HeLa cells (1.0 × 105 cells/mL) were dispersed into 96-well microtiter plates to achieve 200 µL/well. Plates were maintained at 37 °C in 5% CO2/95% air incubator for 24 h. The cells in each well were incubated with 25 µL probe for various periods or various probe concentrations at a fixed time. Subsequently, 20 µL of 5 mg/mL MTT (3-(4,5-dimethythiazol-2-yl)2,5-diphenyl tetrazolium bromide) in PBS was added into each well, followed by further incubation at 37 °C for 4 h. Then, 100 µL of dimethyl sulphoxide was added to each well and incubated under circular shaking for 15 min to dissolve the precipitates. The absorbance was measured at 490 nm using a microplate reader (680, Bio-Rad, USA).

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Detection of Telomerase Activity. Different volumes of cell lysates were incubated with a mixture containing of 50 µL probe (6 nM), 20 µL 10× TRAP reaction buffer and 4 µL 10 mM dNTPs at 37 °C for 2 h (in all test, the final volumes were adjusted to 200 µL with DEPC-treated water). The fluorescence spectra of the products was measured on a fluorophotometer with λex of 633 nm. For control experiments, telomerase in the cell lysates was deactivated by heating at 90 °C for 30 min. PCR Amplification and Gel Electrophoresis. The reaction for the elongation of TP end with the telomeric repeat TTAGGG was performed using cell lysates or telomerase. 2 µL of cell lysate or telomerase solution was mixed with 3 µL TP-carrying strand, 16 µL PCR water, 2.5 µL 10× TRAP reaction buffer, 0.5 µL 50× dNTP mix, 0.5 µL TRAP primer mixture, and 0.5 µL Taq polymerase (5 units/µL) in a centrifuge tube, the solution was incubated in water bath at 37 °C for 30 min to extend the TP primer. The final TP strands with (TTAGGG)n were amplified with PCR (32 cycles, 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min). For gel electrophoresis, samples were mixed with 2.0 µL loading buffer, and loaded onto polyacrylamide hydrogel. Electrophoresis was carried out in trisborate-EDTA (TBE) buffer at 110 V for 65 min. The gel was stained for 10 min with 4S Red Plus and imaged using with a Tocan 240 gel imaging system (Shanghai Tocan Biotechnology Company). Fluorescence Microscopy and Flow Cytometry. Cells (1.0 × 105 cell/mL) were seeded onto glassbottom confocal dishes and cultured for 24 h. SNAB probes (50 µL) were added into each confocal dish and incubated for different time periods. In the case of control experiments, telomerase inhibitor AZT was first co-incubated with cells for 48 h before the addition of probes. Fluorescent imaging was conducted on a Zeiss confocal microscope (LSM 710) with the excitation of 633 nm and the collection at the window of 640-750 nm. Flow cytometry analysis was performed on a Cytoflex flow cytometer (Beckman), using the APC-A fluorescence channel (excitation 638nm). Data was collected for approximately 10,000 cells per sample. Tumor Models and in Vivo Imaging. Four-week old athymic nude mice were purchased from Nanjing Biomedical Research Institute (Nanjing University, China), and maintained in pathogen-free ACS Paragon Plus Environment

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facilities. To establish human cervix tumor in mice, one million HeLa cells suspended in 100 µL PBS were mixed with 100 µL matrigel (BD Biosciences) and injected subcutaneously near the armpit area. The HeLa-implanted mice were maintained until the size of tumors reached ~300 mm3. Tumor volumes were calculated (W2 × L)/2 in which width (W) is defined as the shorter dimension of two measurements and length (L) is defined as the longer one. Following similar methods, A549 and Hep G2 cells were also subcutaneously implanted under the armpit of athymic nude mice to establish lung and liver tumor models, respectively. For intratumor injection, 100 µL of the SNAB probe was administered, and mice were anesthetized. Fluorescence imaging of tumors in the nude mouse was performed at different time intervals on an IVIS Lumina LT in vivo imaging system (Caliper Life Sciences, excitation of 640 nm, emission of 650-750 nm). Frozen Sections of Tumors. Tumors removed from mice were fixed in 4% formalin for 24 h. Tissue sections were cut on a cryostat (Leica CM1510 S). The sections were treated with 200 µL DAPI (50 µg/mL) for 20 min, and washed with PBS buffer six times. After the sections were treated with dimethylbenzene for 15 min, one or two drops of mounting medium were added prior to sealing with a cover slip. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Acknowledgement This work was supported in part by the National Basic Research Program of China (2015CB932002), National Natural Science Foundation of China (21335006, 21475135, 21775001, 61705239, 21605145, 21705001), and Natural Science Foundation of Anhui Province in China (1608085QB32). AUTHOR INFORMATION ACS Paragon Plus Environment

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Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

Z. Liu and J. Zhao contributed equally to this work.

Author Contributions This work was achieved through an international collaboration among Z.Z. M.H. and X.G.’s labs. Z.L., J.Z., Z.Z., M.H. and X.G. conceived the idea, designed the experiments, and wrote the paper. Z.Z. also supervised the day-to-day operation. Z.L. prepared the probe and performed majority of the in vitro assays. J.Z. and G.H. together did the cell studies. R.Z., J.Z. and C.Z. did the in vivo experiments. R.Z., J.Z. and B.L. helped analyze and interpret the data. REFERENCES (1) Navin, N.; Kendall, J.; Troge, J.; Andrews, P.; Rodgers, L.; McIndoo, J.; Cook, K.; Stepansky, A.; Levy, D.; Esposito, D.; Muthuswamy, L.; Krasnitz, A.; McCombie, W. R.; Hicks, J.; Wigler, M. Tumour Evolution Inferred by Single-Cell Sequencing. Nature 2011, 472, 90-94. (2) Hajitou, A.; Trepel, M.; Lilley, C. E.; Soghomonyan, S.; Alauddin, M. M.; Marini, F. C., 3rd; Restel, B. H.; Ozawa, M. G.; Moya, C. A.; Rangel, R.; Sun, Y.; Zaoui, K.; Schmidt, M.; von Kalle, C.; Weitzman, M. D.; Gelovani, J. G.; Pasqualini, R.; Arap, W. A Hybrid Vector for Ligand-Directed Tumor Targeting and Molecular Imaging. Cell 2006, 125, 385-398. (3) Arlt, C.; Flegler, V.; Ihling, C. H.; Schafer, M.; Thondorf, I.; Sinz, A. An Integrated Mass Spectrometry Based Approach to Probe the Structure of the Full-Length Wild-Type Tetrameric p53 Tumor Suppressor. Angew. Chem. Int. Ed. 2017, 56, 275-279. (4) Tsujikawa, T.; Kumar, S.; Borkar, R. N.; Azimi, V.; Thibault, G.; Chang, Y. H.; Balter, A.; ACS Paragon Plus Environment

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Kawashima, R.; Choe, G.; Sauer, D.; El Rassi, E.; Clayburgh, D. R.; Kulesz-Martin, M. F.; Lutz, E. R.; Zheng, L.; Jaffee, E. M.; Leyshock, P.; Margolin, A. A.; Mori, M.; Gray, J. W.; Flint, P. W.; Coussens, L. M. Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis. Cell Rep. 2017, 19, 203-217. (5) McClintock, B. The Stability of Broken Ends of Shromosomes in Zea Mays. Genetics. 1941, 26, 234-282. (6) Blackburn, E. H. Telomeres: Do the Ends Justify the Means. Cell 1984, 37, 7-8. (7) Levis, R. W. Viable Deletions of a Telomere from a Drosophila Chromosome. Cell 1989, 58, 791801. (8) De Lange, T. How Telomeres Solve the End-Protection Problem. Science 2009, 326, 948-952. (9) Yu, G. L.; Bradley, J. D.; Attardi, L. D.; Blackburn, E. H. In Vivo Alteration of Telomere Sequences and Senescence Caused by Mutated Tetrahymena Telomerase RNAs. Nature 1990, 344, 126-132. (10) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Specific Association of Human Telomerase Activity with Immortal Cells and Cancer. Science 1994, 266, 2011-2015. (11) Akincilar, S. C.; Unal, B.; Tergaonkar, V. Reactivation of Telomerase in Cancer. Cell. Mol. Life Sci. 2016, 73, 1659-1670. (12) Harley, C. B. Telomerase and Cancer Therapeutics. Nat. Rev. Cancer 2008, 8, 167-179. (13) Shay, J. W.; Wright, W. E. Senescence and Immortalization: Role of Telomeres and Telomerase. Carcinogenesis 2005, 26, 867-874. (14) Shay, J. W.; Bacchetti, S. A Survey of Telomerase Activity in Human Cancer. Eur. J. Cancer 1997, 33, 787-791. (15) Yang, C.; Przyborski, S.; Cooke, M. J.; Zhang, X.; Stewart, R.; Anyfantis, G.; Atkinson, S. P.; Saretzki, G.; Armstrong, L.; Lako, M. A Key Role for Telomerase Reverse Transcriptase Unit in Modulating Human Embryonic Stem Cell Proliferation, Cell Cycle Dynamics, and in Vitro ACS Paragon Plus Environment

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Differentiation. Stem Cells 2008, 26, 850-863. (16) Gammaitoni, L.; Weisel, K. C.; Gunetti, M.; Wu, K. D.; Bruno, S.; Pinelli, S.; Bonati, A.; Aglietta, M.; Moore, M. A.; Piacibello, W. Elevated Telomerase Activity and Minimal Telomere Loss in Cord Blood Long-Term Cultures with Extensive Stem Cell Replication. Blood 2004, 103, 4440-4448. (17) Pech, M. F.; Garbuzov, A.; Hasegawa, K.; Sukhwani, M.; Zhang, R. X. J.; Benayoun, B. A.; Brockman, S. A.; Lin, S. D.; Brunet, A.; Orwig, K. E.; Artandi, S. E. High Telomerase Is a Hallmark of Undifferentiated Spermatogonia and Is Required for Maintenance of Male Germline Stem Cells. Gene. Dev. 2015, 29, 2420-2434. (18) Xiao, Y.; Dane, K. Y.; Uzawa, T.; Csordas, A.; Qian, J.; Soh, H. T.; Daugherty, P. S.; Lagally, E. T.; Heeger, A. J.; Plaxco, K. W. Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-Modified Gold Nanoparticles. J. Am. Chem. Soc. 2010, 132, 15299-15307. (19) Tian, L.; Weizmann, Y. Real-Time Detection of Telomerase Activity Using the Exponential Isothermal Amplification of Telomere Repeat Assay. J. Am. Chem. Soc. 2013, 135, 1661-1664. (20) Qian, R.; Ding, L.; Ju, H. Switchable Fluorescent Imaging of Intracellular Telomerase Activity Using Telomerase-Responsive Mesoporous Silica Nanoparticle. J. Am. Chem. Soc. 2013, 135, 1328213285. (21) Qian, R.; Ding, L.; Yan, L.; Lin, M.; Ju, H. A Robust Probe for Lighting Up Intracellular Telomerase via Primer Extension to Open a Nicked Molecular Beacon. J. Am. Chem. Soc. 2014, 136, 8205-8208. (22) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-Based Method for Rationally Assembling Nanoparticles into Macroscopic Materials. Nature 1996, 382, 607-609. (23) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134, 1376-1391. (24) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. Nano-Flares: Probes for Transfection and mRNA Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 15477-15479. ACS Paragon Plus Environment

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(25) Narayan, S. P.; Choi, C. H.; Hao, L.; Calabrese, C. M.; Auyeung, E.; Zhang, C.; Goor, O. J.; Mirkin, C. A. The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates. Small 2015, 11, 4173-4182. (26) Massich, M. D.; Giljohann, D. A.; Seferos, D. S.; Ludlow, L. E.; Horvath, C. M.; Mirkin, C. A. Regulating Immune Response Using Polyvalent Nucleic Acid-Gold Nanoparticle Conjugates. Mol. Pharmaceut. 2009, 6, 1934-1940. (27) Sita, T. L.; Kouri, F. M.; Hurley, L. A.; Merkel, T. J.; Chalastanis, A.; May, J. L.; Ghelfi, S. T.; Cole, L. E.; Cayton, T. C.; Barnaby, S. N.; Sprangers, A. J.; Savalia, N.; James, C. D.; Lee, A.; Mirkin, C. A.; Stegh, A. H. Dual Bioluminescence and Near-Infrared Fluorescence Monitoring to Evaluate Spherical Nucleic Acid Nanoconjugate Activity in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 41294134. (28) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; Mirkin, C. A. Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles. Nano Lett. 2007, 7, 3818-3821. (29) Greider, C. W.; Blackburn, E. H. Identification of a Specific Telomere Terminal Transferase Activity in Tetrahymena Extracts. Cell 1985, 43, 405-413. (30) Blackburn, E. H. Telomeres and Their Synthesis. Harvey. Lect. 1990, 86, 1-18. (31) Morin, G. B. The Human Telomere Terminal Transferase Enzyme Is a Ribonucleoprotein That Synthesizes TTAGGG Repeats. Cell 1989, 59, 521-529. (32) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations. J. Am. Chem. Soc. 2015, 137, 8340-8343. (33) Ponnuswamy, N.; Bastings, M. M. C.; Nathwani, B.; Ryu, J. H.; Chou, L. Y. T.; Vinther, M.; Li, W. A.; Anastassacos, F. M.; Mooney, D. J.; Shih, W. M. Oligolysine-Based Coating Protects DNA Nanostructures from Low-Salt Denaturation and Nuclease Degradation. Nat. Commun. 2017, 8, 15654ACS Paragon Plus Environment

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15662. (34) Probst, C. E.; Zrazhevskiy, P.; Gao, X. H. Rapid Multitarget Immunomagnetic Separation through Programmable DNA Linker Displacement. J. Am. Chem. Soc. 2011, 133, 17126-17129. (35) Zhang, D. Y.; Seelig, G. Dynamic DNA Nanotechnology Using Strand-Displacement Reactions. Nat. Chem. 2011, 3, 103-113. (36) Zheng, D.; Giljohann, D. A.; Chen, D. L.; Massich, M. D.; Wang, X. Q.; Iordanov, H.; Mirkin, C. A.; Paller, A. S. Topical Delivery of siRNA-Based Spherical Nucleic Acid Nanoparticle Conjugates for Gene Regulation. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11975-11980. (37) Khan, A.; Benboubetra, M.; Sayyed, P. Z.; Ng, K. W.; Fox, S.; Beck, G.; Benter, I. F.; Akhtar, S. Sustained Polymeric Delivery of Gene Silencing Antisense ODNs, siRNA, DNAzymes and Ribozymes: in Vitro and in Vivo Studies. J. Drug. Target. 2004, 12, 393-404. (38) Alhasan, A. H.; Kim, D. Y.; Daniel, W. L.; Watson, E.; Meeks, J. J.; Thaxton, C. S.; Mirkin, C. A. Scanometric microRNA Array Profiling of Prostate Cancer Markers Using Spherical Nucleic AcidGold Nanoparticle Conjugates. Anal. Chem. 2012, 84, 4153-4160. (39) Zhang, K.; Hao, L.; Hurst, S. J.; Mirkin, C. A. Antibody-Linked Spherical Nucleic Acids for Cellular Targeting. J. Am. Chem. Soc. 2012, 134, 16488-16491. (40) Radovic-Moreno, A. F.; Chernyak, N.; Mader, C. C.; Nallagatla, S.; Kang, R. S.; Hao, L.; Walker, D. A.; Halo, T. L.; Merkel, T. J.; Rische, C. H.; Anantatmula, S.; Burkhart, M.; Mirkin, C. A.; Gryaznov, S. M. Immunomodulatory Spherical Nucleic Acids. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 3892-3897. (41) Sharma, H. W.; Sokoloski, J. A.; Perez, J. R.; Maltese, J. Y.; Sartorelli, A. C.; Stein, C. A.; Nichols, G.; Khaled, Z.; Telang, N. T.; Narayanan, R. Differentiation of Immortal Cells Inhibits Telomerase Activity. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 12343-12346. (42) Rha, S. Y.; Park, K. H.; Kim, T. S.; Yoo, N. C.; Yang, W. I.; Roh, J. K.; Min, J. S.; Lee, K. S.; Kim, B. S.; Choi, J. H.; Lim, H. Y.; Chung, H. C. Changes of Telomerase and Telomere Lengths in Paired ACS Paragon Plus Environment

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Synopsis: Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem ACS Paragon Plus Environment

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of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms, ranging from solution-based assay, to single cell imaging and in vivo solid tumor imaging.

Figure 1. Schematic of the SNAB technology and telomerase detection in solution. (A) SNAB probe structure and working mechanism. Fluorescence of the TP-carrying strand and FL-strand duplex on AuNPs surface is initially quenched. Telomerase activity leads to extension of the TP-carrying strand with telomeric repeat (TTAGGG). Formation of a more stable hairpin structure displaces the FL-strand, restoring fluorescence. (B) SNAB cell entry for imaging telomerase activity in live cells. (C) Fluorescent spectra of SNAB probes after incubation with various dosages of telomerase (left). Fluorescence enhancement factor plotted against telomerase dosage (right). (D) Fluorescent spectra of

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SNAB probes after incubation with HeLa cell lysates containing different cell numbers (left). Fluorescence enhancement factor plotted against cell numbers (right). The insets in C and D are the representative fluorescent photograph excited with 633 nm laser. The error bars represent standard deviation ± SD from three tests.

Figure 2. Probe optimization and verification of the detection mechanism. Five FL-strands of different length (8, 11, 12, 13 and 18 bases) complementary to the TP-carrying strands were tested. (A) Melting temperatures of the probes. Insets are fluorescent photographs of the probe solution at 37 °C excited with a 633 nm laser. (B) Fluorescent intensities of the probes with or without telomerase at 37 °C. (C) Gel electrophoresis of PCR-amplified TP-carrying strand after exposure to telomerase or cell lysates (tumor cell: HeLa; normal somatic cell: MRC-5). (D) Gel electrophoresis verifying the proposed ACS Paragon Plus Environment

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working principle. The duplex beacon (without AuNPs) after exposure to telomerase or HeLa lysate show band shifts in contrast to after exposure to lysis buffer or MRC-5 lysate, (left) DNA bands stained with 4S Red Plus®; (right) the corresponding fluorescent image from the FL-strand. The green fluorophore in the FL-strand was taken with long-wavelength UV excitation.

Figure 3. Fluorescent imaging of telomerase activity in live cells (excitation 633 nm, emission 640-750 nm). (A) Dosage dependent fluorescent images of HeLa cells treated with the SNAB probe for 2 h. (B) Time dependent fluorescent images of HeLa cells incubated with the SNAB probe (6 nM). (C) Control experiments by addition of different amounts of telomerase inhibitor, 3′-Azido-3′-deoxythymidine (AZT) (upper: bright field; bottom: fluorescent mode).

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Figure 4. Telomerase activity in various cell lines characterized with fluorescent microscopy and flow cytometry. (A) Fluorescent micrographs of 10 common tumor cells treated with the SNAB probe. Cell nucleus is counter-stained with 4',6-diamidino-2-phenylindole (DAPI). (B) Mean fluorescent enhancement factors of the cells before and after the SNAB probe treatment. (C) Average fluorescent intensities obtained from over 10,000 cells per cell line after SNAB probe treatment by flow cytometry (inset: representative histograms from HeLa cells and MRC-5 cells).

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Figure 5. In vivo imaging of telomerase activities in solid tumors. (A) Time-lapse fluorescent images of tumor in vivo after SNAB administration. (Red circle) tumor and (black circle) tumor free tissue. Fluorescence signals only turn on in the solid tumors. In vivo images were obtained with excitation wavelength of 640 nm and emission wavelength of 650-750 nm. (B) Confocal images of tumor section (5 µm thick) from probe-injected nude mice (red fluorescence). The sections are counter-stained with DAPI for cell nuclei (blue).

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