Simultaneous Fluorescence Visualization of EpithelialâMesenchymal

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Simultaneous Fluorescence Visualization of EMT and Apoptosis Processes in Tumor Cells for Evaluating the Impact of EMT on Drug Efficacy Mingming Luan, Jinjie Chang, Wei Pan, Yuanyuan Chen, Na Li, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02494 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Analytical Chemistry

Simultaneous Fluorescence Visualization of EMT and Apoptosis Processes in Tumor Cells for Evaluating the Impact of EMT on Drug Efficacy Mingming Luan, Jinjie Chang, Wei Pan, Yuanyuan Chen, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China. ABSTRACT: The epithelial-mesenchymal transition (EMT) process plays a pivotal role in acquiring invasive and metastatic properties and has been recognized as a crucial driver of epithelial-derived tumor malignancies. It is necessary to determine the role of EMT in promoting or suppressing carcinoma progression through investigating the relationship between EMT and apoptosis. We designed a multicolor fluorescent nanoprobe for simultaneously imaging the epithelial biomarker E-cadherin mRNA, the mesenchymal marker vimentin mRNA and the apoptotic marker caspase-3. EMT and apoptosis progresses could be visually detected, which were used to study the effect of EMT on apoptosis and further assess the influence of EMT on drug efficacy in different cancer cells. We believe the designed nanoprobe can offer a new strategy for visualizing EMT and apoptosis in tumor cells and will be a promising tool to investigate the efficiency of drugs targeting EMT-related therapies in living cells.

Malignant tumors are a highly fatal disease; the primary cause of poor prognosis and death is tumor cell invasion and metastasis.1-4 Tumor progression toward invasion and metastasis is a stepwise and multistage process and the epithelial-mesenchymal transition (EMT) process is essential for invasive and metastatic properties.5-7 EMT is involved in development, fibrosis, homeostasis, cancer progression and wound healing. EMT can be promoted by many inducers, including TGF-β (transforming growth factor-β), hypoxia and EGF (epidermal growth factor). The E-cadherin mRNA (an epithelial biomarker gene) expression decreases, while the vimentin mRNA (a mesenchymal biomarker gene) expression increases during EMT progression.8-11 The loose cell-cell adhesion, enhanced motility and elongated spindle-like morphologies during EMT result in more migratory and invasive traits and exhibit stronger resistance to antitumor drugs and apoptosis.12-13 Many reports have shown that the process of EMT is associated significantly with the invasion and metastasis of epithelial-derived malignant tumors, such as breast, pancreatic and colorectal carcinomas.14-16 Cancer therapeutics that target EMT progress could reduce drug resistance, invasion and anti-apoptosis through regulating EMT-related genes, growth factors and signaling pathways. Hence, targeting EMT in cancer therapy is a promising avenue of great interest.17-22 Apoptosis and EMT are usually considered as separate processes because EMT is closely related to the enhanced anti-apoptotic capacity. However, recent studies

have found that a subset of cancer cells can undergo a lethal EMT resulting in cell apoptosis and tumor suppression.23-26 Therefore, it is important to determine whether EMT will promote or suppress cancer progress by studying the relationship between EMT and apoptosis. Currently, many methods have been applied to detect EMT and apoptosis processes. Real-time reverse transcription-PCR (RT-PCR) and western blot (WB) analyses are biological techniques based on biomarkers, but these approaches have disadvantages of being cumbersome to operate and being time consuming. In addition, the cells are treated with organic solvent, and the samples are measured directly in cell homogenates.27-28 Fluorescent probes have been applied to detect EMT markers or apoptosis-related markers in living cells, but these probes are used to specifically monitor EMT or apoptosis markers.29-31 Therefore, it is necessary to design a single probe that can simultaneously monitor EMT and apoptosis progresses in living cells. Hence, we designed and synthesized a fluorescent nanoprobe to simultaneously detect E-cadherin mRNA, vimentin mRNA and caspase-3. The nanoprobe was formulated by gradually modified the gold nanoparticles (AuNPs) with two molecular beacons (MBs) and a peptide (Scheme 1).32-36 Generally, the fluorescence of three dye molecules connected with a peptide and two MBs is well extinguished by AuNPs. The MBs could specifically respond to E-cadherin mRNA and vimentin mRNA targets, and the peptide chain was cleaved by caspase-3, generating the three fluorophores’ fluorescent signals.

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To our knowledge, such a nanoprobe opens up new avenue to simultaneously detect the processes of EMT and apoptosis in living cells. Scheme 1. Simultaneous detection of EMT and apoptosis in living cells using the fluorescent nanoprobe based on AuNPs.

EXPERIMENTAL SECTION Intracellular Imaging of the Nanoprobe. MCF-10A (human noncancerous breast cell), MDA-MB-231 (human breast cancer cell line) and PANC-1 (human pancreatic cancer cell line) were purchased from Procell (Wuhan, China). MCF-7 (human breast carcinoma cell line) were seeded in confocal dishes and cultured for 24 h. The four different cells were then treated with the fluorescent nanoprobe (1 nM) at 37 ℃ for 4 h, the intracellular fluorescent imaging of the cells were then carried out using confocal laser scanning microscopy (CLSM) with three excitation wavelengths (488, 561 and 633 nm); the range of corresponding emission wavelength was 500-550 nm, 575-620 nm, 650-700 nm, respectively. In the experiments for imaging E-cadherin mRNA, vimentin mRNA and caspase-3 in living cells, we choose MCF-10A epithelial cells and MDA-MB-231 mesenchymal cells and divided the cells into two groups. For MCF-10A, one group without TGF-β treatment served as the control, and another group was treated with 5 ng/mL TGF-β for 72 h to induce EMT. 37 Confocal imaging of MCF-10A cells was monitored by CLSM with appropriate laser transmitters after culturing with the fluorescent nanoprobe. For MDA-MB-231, one group without curcumin treatment served as blank, and another group was treated with 10 µM curcumin for 3 days to induce apoptosis.38-39 After incubating with nanoprobe for 4 h, the cellular imaging was performed using CLSM. In the experiments for visualizing the relationship between EMT and apoptotic processes, MCF-7 cells and PANC-1 cells were selected and then sorted into two different groups: an induction group treated with 5 ng/mL TGF-β for 72 h to induce EMT and a control group without treatment. After incubating with the nanoprobe, the cells were then monitored with CLSM. To further investigate EMT and apoptotic processes, PANC1 cells were separated into five parallel groups and treated with 5 ng/mL TGF-β for 0, 18, 36, 48, 72 h, respectively. After treating with the nanoprobe, fluorescent imaging of the cells was performed for the five groups. In the experiments for evaluating the effect of EMT

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process on drug efficacy in different cancer cells, MCF-7 cells and PANC-1 cells were then sorted into five groups in parallel. Group I was the untreated control group; Group II and Group III were treated with 5 ng/mL TGFβ and 10 µM curcumin for 3 days, respectively; Group IV was pretreated with 5 ng/mL TGF-β for 3 days, then curcumin (10 µM) was added for another 3 days; and Group V was pretreated with TGF-β inhibitor SB505124 (2.5 µM) for 1 day,40 then TGF-β (5 ng/mL) was added for 3 days, and finally 10 µM curcumin was added for another 3 days. After all five groups were cultured with the nanoprobe, the CLSM image was then measured. Animal Experiments and Tumor Therapy. All animal experiments were managed in accordance with the Principles of Laboratory Animal Care (People’s Republic of China). MCF-7 breast cancer xenografts were established in 4-week-old female nude mice.41 MCF-7 cells were sorted into five parallel groups. Group I and Group III were not treated; Group II and Group IV were pretreated with 5 ng/mL TGF-β for 3 days; Group V was pretreated with SB505124 (2.5 µM) for 1 day, then TGF-β (5 ng/mL) was added for 3 days. After pretreatment with the corresponding drugs, the five groups of cells (approximately 2 × 106) were then subcutaneously injected into the right sides of five groups of mice, respectively. When the tumor’s volume reached about 100 mm3, five groups of mice were treated as follows: Group I injected with untreated cells served as control and Group II was given intraperitoneal (ip) injection of vehicle (DMSO); Group III-V were given an ip injection of curcumin (0.1 g/kg). The mice were injected once daily for four consecutive days. The body weight and the tumor volume were measured every other day for 14 days. For PANC-1 pancreatic carcinoma xenografts, PANC-1 cells were also separated into five parallel groups: Group I and Group III were only incubated with culture medium without drug treatment; Group II and Group IV were cultured with TGF-β (5 ng/mL) for 3 days; The cells pretreated with 2.5 µM SB505124 for 1 day and further incubated with 5 ng/mL TGF-β for 3 days were served as Group V. The above five groups of cells were inoculated subcutaneously into the right flanks of mice with a suspension of 2 × 106 cells until the tumor volume was reached to 100 mm3. Then, the five groups of mice were subjected to different treatments: Group I, without treatment; Group II, with DMSO; Group III-V, with 0.1 g/kg curcumin. The mice were injected once-daily for 4 days. The mouse weight and tumor size were continuously recorded every two days for two weeks. RESULTS AND DISCUSSION Synthesis and Characterizations of the Fluorescent Nanoprobe. The 20 nm AuNPs were employed in our design, because they have efficient fluorescence quenching ability and can accommodate more recognition units. As shown in Figure S1, AuNPs exhibited a spherical morphology and good dispersion, with approximately 20 nm diameter. No changes of the morphology and dispersion of the nanoprobe were observed after the


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Analytical Chemistry

Figure 1. Fluorescence response of the nanoprobe at the indicated concentrations of: E-cadherin mRNA target (a), vimentin mRNA target (b) and caspase-3 (c). The fluorescent intensities were detected with three excitation wavelengths (488, 561 and 633 nm).

AuNPs were modified with MBs and peptide. The UVVis spectroscopy (Figure S2) showed that the maximum absorption peak of AuNPs was 519 nm, while that of the nanoprobe was 524 nm. The redshift of the maximum absorption indicated that the MBs and peptide were successfully assembled on the surface of the AuNPs. Dynamic light scattering (DLS) and zeta potential were further used to verify the successful preparation of the nanoprobe. Figure S3 indicated that the hydrodynamic diameter changed from 21.6 nm to 27.8 nm. As shown in Figure S4, the zeta potentials of AuNPs and the nanoprobe were -0.171 and -0.816 mV. All these experiments demonstrated that the nanoprobe was successfully synthesized. According to the previously reported protocol,35 each AuNP was calculated to carry 19 ± 1 MBs labeled with ROX targeting E-cadherin mRNA, 21 ± 1 MBs labeled with Cy5 targeting vimentin mRNA, and 77 ± 1 peptides labeled with RhB targeting caspase-3 using the standard curves of the three dyes (Figure S5). In Vitro Investigations of the Nanoprobe. The feasibility of the nanoprobe to identify the caspase-3 target and the two DNA targets was first investigated. Figure 1 showed that the fluorescent signal of each dye molecular was enhanced with increasing concentration of respective target, indicating that the nanoprobe could respond to the specific target. Dynamic experiments of the nanoprobe were further performed. The results showed that the nanoprobe could quickly respond to the caspase-3 target within 2030 min and the two DNA targets within 15-20 min (Figure S6). The matching response time for all three targets indicated that the nanoprobe was suitable for simultaneous detection of these two different target molecules in living cells. Selectivity is an important character for simultaneous detection of different types of target molecules in living cells. Figure S7 showed that the nanoprobe could generate notable fluorescence encountering caspase-3 or the perfectly matched DNA targets. However, the fluorescent signals were weak upon meeting other targets. All the results suggest that the nanoprobe possesses notable specificity and can sense different targets in living cells. MTT Assay. MTT assay was further performed to investigate the cytotoxicity of the nanoprobe. The naked AuNPs (1 nM) and the nanoprobe (1 nM) were incubated with MCF-10A, MCF-7 and PANC-1 cells for 4h, 12h, and 24h, respectively. The results showed that the cell viabil

Figure 2. Confocal imaging of E-cadherin mRNA, vimentin mRNA and caspase-3 in MCF-10A and MDA-MB-231 cells. MCF-10A cells were treated with or without TGF-β and MDA-MB-231 cells were treated with or without curcumin. The cells were cultured with nanoprobe (1 nM) for 4 h at 37 °C. The green, yellow and red channels were recorded with 488, 561 and 633 nm laser, respectively. Scale bars: 100 µm.

ities of the three cell lines were all higher than 90% at different time, which demonstrated that the AuNPs and the nanoprobe both showed little toxicity in living cells (Figure S8). Effect of Curcumin on Cell Proliferation and Apoptosis. We investigated the effect of curcumin concentrations on the proliferation of MDA-MB-231, MCF-7 and PANC-1 cells using Cell Counting Kit-8 (CCK-8). Three cell lines were treated with different concentrations of curcumin (0-50 µM) and the absorbance at 450 nm was measured. The dose curve (Figure S9) showed that curcumin inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner. When the curcumin concentration was more than 20 µM, the cell proliferation was seriously inhibited. Therefore, 10 µM curcumin was chosen in the following experiments. Fluorescence Imaging of Intracellular EMT and Apoptosis Processes. To investigate the capability of the nanoprobe to concurrently monitor EMT and apoptosis processes, two cell lines were selected: MCF-10A and MDA-MB-231. MCF-10A was chosen to assess the ability of the nanoprobe for detecting EMT process because EMT could be induced in this cell line treated with TGF-β.37 MDA-MB-231 was chosen to evaluate the feasibility of the nanoprobe for detecting apoptosis process because curcumin could induce apoptosis in MDAMB-231 cell line.38-39 MCF-10A cells were divided into two groups and analyzed in parallel. One group was treated with TGF-β to promote the progress of EMT, and the other group without treatment served as the



blank. After culturing with the nanoprobe, the cells were monitored by CLSM. Figure 2 showed that the yellow fluorescent signal for E-cadherin mRNA was weaker and that the red fluorescent signal for vimentin mRNA was stronger in cells treated with TGF-β compared to the untreated cells. This finding indicated that MCF-10A cells underwent an EMT, accompanied by a decrease of E-cadherin mRNA and an increase of vimentin mRNA. The bright-field images showed that MCF-10A cells treated with TGF-β had an elongated, spindle-like morphology demonstrating that the cells experienced EMT after TGF-β treatment. The results of the RT-PCR (Figure S10) maintained consistency with the results of CLSM, implying that the prepared nanoprobe could visually detect EMT progress in living cells. In MCF-10A cells treated with TGF-β, the green fluorescence for caspase-3 was not observed, which was confirmed by WB results (Figure S12), suggesting that no apoptotic process appeared during EMT. MDA-MB-231 cells were also sorted into two parallel groups: a curcumin-treated group and a control group without treatment. The cells were then cultured with the nanoprobe. Figure 2 showed that the yellow fluorescent signal was low and the red fluorescent signal was high in the control group indicating that MDA-MB-231 cells were positive for vimentin mRNA and negative for E-cadherin mRNA. Strong green fluorescent signal for caspase-3 was observed in the curcumin-treated group, which suggested that curcumin induced cell apoptosis. The changes in abundance of caspase-3, vimentin mRNA and E-cadherin mRNA were confirmed by the results of RT-PCR and WB (Figures S11 and S12), which implies that the nanoprobe can be used to visually monitor apoptosis in cells.

Next, the nanoprobe was applied to visually investigate the relationship between EMT and apoptosis. MCF7 and PANC-1 cell lines were chosen and separated into two parallel groups, respectively. One group was treated with TGF-β to induce EMT process and another group without treatment used as blank. The two cells were imaged by CLSM after being cultured with the nanoprobe. Figure 3 showed that the red fluorescent intensity was increased and the yellow fluorescent intensity was decreased in MCF-7 cells treated with TGF-β, which indicated that TGF-β promoted the process of EMT. Meanwhile, the green fluorescence was not observed in MCF-7 cells treated with TGF-β implying that no apoptosis appeared during EMT. The fluorescent imaging of PANC-1 cells was also observed by CLSM. The yellow and red fluorescent changes in PANC-1 cells with TGF-β treatment were similar to that in MCF-7 cells, which revealed that PANC-1 cells also underwent EMT. Interestingly, the strong green fluorescence was monitored in TGF-β-treated PANC-1 cells, indicating that apoptosis appeared during EMT. Moreover, apoptotic bodies could be clearly observed in the bright field. The analyses of RT-PCR (Figures S13 and S14) and WB (Figures S18 and S20) further confirmed the expression levels of the three biomarkers. For investigating the relationship between EMT and apoptosis, PANC-1 cells were further treated with TGF-β for different times and imaged. As shown in Figure 4, the yellow fluorescent intensity gradually decreased and the red fluorescent intensity gradually increased with increasing time of exposure to TGF-β, indicating that EMT progress was induced. The green fluorescent signal began to appear at 36 h and became stronger with increasing time, implying the appearance of apoptosis. The bright-field imaging showed that PANC-1 cells initially maintained an epithelial phenotype but possessed a more spindle-like, elongated phenotype after treatment with TGF-β for 18 hours. From 36-72 h, more apoptotic bodies appeared. These results indicated that

Figure 3. Intracellular imaging of E-cadherin mRNA, vimentin mRNA and caspase-3 in MCF-7 and PANC-1 cells under CLSM. The two cell lines were treated with or without TGF-β. Scale bars: 100 µm.

Figure 4. Imaging of E-cadherin mRNA, vimentin mRNA and caspase-3 in PANC-1 cells treated with TGF-β for different times. Scale bars: 100 µm.


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Analytical Chemistry EMT induced by TGF-β preceded the apoptosis of PANC-1 cells. The results of RT-PCR (Figure S15) and WB (Figure S16) were consistent with the CLSM results, which demonstrates that the nanoprobe can simultaneously detect EMT and apoptosis progresses in cells. Recent studies have shown that the process of EMT is closely related to antitumor drug resistance and a poor curative effect. Therefore, the effect of EMT on drug efficiency in different cancer cells was determined through detecting the fluorescent signals of the three markers. MCF-7 and PANC-1 cells were separated into five parallel groups: Group I without treatment used as control; Group II and Group III were treated with TGF-β and curcumin, respectively; Group IV was pretreated with TGF-β followed by curcumin treatment; and Group V was pretreated with SB505124, followed successively by treatment with TGF-β and curcumin. The cells were cultured with nanoprobe and monitored under CLSM. Fluorescent imaging of MCF-7 cells is shown in Figure 5. Compared with Group I, the relative fluorescent changes of the three markers in Group II indicated that MCF-7 cells underwent EMT but did not experience apoptosis, and the strong green fluorescence in Group III suggests that curcumin induced MCF-7 cell apoptosis. Compared to Group III, the red fluorescent signal slightly increased while the yellow and green fluorescent signals decreased significantly in Group IV; similar fluorescent intensities of the three biomarkers in Group V cells were observed. These results showed that the process of EMT induced by TGF-β conferred resistance to apoptosis mediated by curcumin and decreased drug efficacy, and the antiapoptosis properties were inhibited by the TGF-β inhibitor SB505124. RT-PCR and WB results (Figures S17 and S18) confirmed the relative expressions of the three biomarkers. The results showed that if MCF-7 cells underwent EMT induced by TGF-β, it was necessary to suppress EMT before treatment with curcumin.

We next selected PANC-1 cells and evaluated the influence of EMT induced by TGF-β on the efficacy of drug therapy. The cells were also divided into five groups: Group I, control group without drug treatment; Group II, TGF-β treatment; Group III, curcumin treatment; Group IV, successive treatment with TGF-β and curcumin; Group V, successive treatment with SB505124, TGF-β and curcumin. The fluorescence imagines of five groups of PANC-1 cells are shown in Figure 6. The yellow fluorescent signal for E-cadherin mRNA in Group II was weaker than that in Group I. While the red fluorescent signal for vimentin mRNA and the green fluorescent signal for caspase-3 were stronger. The results showed that PANC-1 cells treated with TGF-β underwent both EMT and apoptosis. The strong green fluorescent signal in Group III indicated that the apoptosis was induced by curcumin. The green fluorescence in Group IV dramatically increased compared to Group III, indicating that EMT enhanced apoptosis caused by curcumin and the drug efficacy of curcumin was improved. For Group V, when the TGF-β-induced EMT was inhibited by SB505124, the green fluorescent signal was weaker than that in Group IV, demonstrating that the drug efficacy of curcumin was decreased without EMT. The results suggested that EMT induced by TGF-β could enhance the drug efficacy of curcumin for PANC-1 cells. The changes in the abundance of vimentin mRNA, Ecadherin mRNA and caspase-3 were further ascertained by RT-PCR and WB analyses (Figures S19 and S20). All these results were consistent with the confocal fluores-

Figure 5. Imaging of E-cadherin mRNA, vimentin mRNA and caspase-3 in MCF-7 cells under CLSM. The cells were treated with TGF-β, curcumin (Cur) or TGF-β inhibitor SB505124 (SB). Scale bars: 100 µm.

Figure 6. Confocal fluorescence images of E-cadherin mRNA, vimentin mRNA and caspase-3 in PANC-1 cells under CLSM. The cells were treated with TGF-β, curcumin or SB505124. Scale bars: 100 µm.

cence imaging results.

Evaluating the Influence of EMT on Drug Efficacy in vivo. To determine the feasibility of the prepared nanoprobe for predicting the influence of EMT on the therapeutic effects in living cells, xenograft models with MCF-7 and PANC-1 cells (pretreated with TGF-β or SB505124, respectively) were selected. The tumor beari-



Figure 7. In vivo evaluation of the effect of EMT on drug efficacy in mice bearing MCF-7 tumor. (a) Photos were taken before treatment (0 day) and on the 14th day posttreatment with different treatments; (b) tumor growth curves and (c) body weight curves were measured at 2-day intervals for 14 days.

ng mice were separated into five parallel groups: control group (DMSO solvent only), TGF-β, curcumin, TGF-β + curcumin, and SB505124 + TGF-β + curcumin. As seen in Figures 7a and 7b, the MCF-7 tumors in the control group and the TGF-β group exhibited an approximately 3-fold increase compared to their initial volumes. The tumor sizes in the curcumin group and the SB505124 + TGF-β + curcumin group significantly decreased. Interestingly, the tumor volumes in the TGF-β + curcumin group were smaller than those in control group and larger than those in the curcumin group. The body weights of all the mice (Figure 7c) had no obvious changes during tumor therapy, implying that these treatments have no obvious toxicity. These results suggest that the process of EMT stimulated by TGF-β can reduce the efficacy of curcumin in MCF-7 tumors. The PANC-1 tumor-bearing mice are shown in Figures 8a and 8b. Compared to the initial tumor volumes, rapid tumor growth was observed in the control group, and slow tumor growth was found in the TGF-β group. The tumor volumes in the curcumin group and the SB505124 + TGF-β + curcumin group were reduced to approximately 50% of their original volumes; interestingly, the tumors in the TGF-β + curcumin group showed an 18% reduction in volume. These results demonstrated that TGF-β-promoted EMT could increase therapeutic efficacy of curcumin and the synergistic therapy based on EMT and curcumin could be a more efficacious approach for the therapy of PANC-1 tumors. Weight changes over time (Figure 8c) showed that the treatments had no obvious toxicity. The results of the in vivo experiments confirmed the results of intracellular imaging, indicating that the nanoprobe could be successfully applied to evaluate the effect of EMT on drug efficacy through simultaneously detecting of EMT and apoptosis in living cells.

Figure 8. In vivo evaluation of the effect of EMT on drug efficacy in mice bearing PANC-1 tumor. (a) Photos were taken before treatment (0 day) and on the 14th day posttreatment with different treatments; (b) tumor growth curves and (c) body weight curves were measured at 2-day intervals for 14 days.

CONCLUSIONS In summary, a multicolor fluorescent nanoprobe was designed to successfully visualize EMT and apoptosis processes by simultaneously detecting the EMT epithelial biomarker E-cadherin mRNA, the mesenchymal biomarker vimentin mRNA and the apoptotic marker caspase-3 in living cells. The nanoprobe exhibited high specificity, excellent biocompatibility and a rapid response. The results of live-cell imaging and in vivo experiments indicated that the nanoprobe could be used to visually evaluate the relationship between EMT and apoptosis in different tumor cells and to further assess the effect of EMT on drug efficiency. The prepared multicolor nanoprobe can offer a new perspective on predicting the feasibility of EMT-targeted therapy at the cellular level.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Fax: (86)-531-86180017. E-mail: [email protected]. *Fax: (86)-531-86180017. E-mail: [email protected].

ORCID Na Li: 0000-0002-0392-6672 Bo Tang: 0000-0002-8712-7025

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS


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Analytical Chemistry This work was supported by National Natural Science Foundation of China (21535004, 91753111, 21775094, 21505087, 21390411) and the Key Research and Development Program of Shandong Province (2018YFJH0502).

REFERENCES (1) Friedl, P.; Alexander, S. Cell 2011, 147, 992-1009. (2) Slattum, G. M.; Rosenblatt, J. Nat. Rev. Cancer 2014, 14, 495501. (3) Hsieh, A. C.; Liu, Y.; Edlind, M. P.; Ingolia, N. T.; Janes, M. R.; Sher, A.; Shi, E. Y.; Stumpf, C. R.; Christensen, C.; Bonham, M. J.; Wang, S.; Ren, P.; Martin, M.; Jessen, K.; Feldman, M. E.; Weissman, J. S.; Shokat, K. M.; Rommel, C.; Ruggero, D. Nature 2012, 485, 55-61. (4) Chaffer, C. L.; Weinberg, R. A. Science 2011, 331, 1559-1564. (5) Jin, K.; Li, T.; Dam, H.; Zhou, F.; Zhang, L. J. Pathol. 2017, 241, 567–577. (6) Oskarsson, T.; Batlle, E.; Massague, J. Cell Stem Cell 2014, 14, 306–321. (7) Valastyan, S.; Weinberg, R. A. Cell 2011, 147, 275–292. (8) Thiery, J. P.; Acloque, H.; Huang, R. Y.; Nieto, M. A. Cell 2009, 139, 871-890. (9) Wilson, C.; Nicholes, K.; Bustos, D.; Lin, E.; Song, Q.; Stephan, J.; Kirkpatrick, D. S.; Settleman, J. Oncotarget 2014, 5, 7328–7341. (10) Buonato, J. M.; Lazzara, M. J. Cancer Res. 2014, 74, 309–319. (11) De Craene, B.; Berx, G. Nat. Rev. Cancer 2013, 13, 97-110. (12) Nieto, M. A.; Huang, R. Y.; Jackson, R. A.; Thiery, J. P. Cell 2016, 166, 21-45. (13) Tam, W. L.; Weinberg, R. A. Nat. Med. 2013, 19, 1438-1449. (14) Rhim, A. D.; Mirek, E. T.; Aiello, N. M.; Maitra, A.; Bailey, J. M.; McAllister, F.; Reichert, M.; Beatty, G. L.; Rustgi, A. K.; Vonderheide, R. H.; Leach, S. D.; Stanger, B. Z. Cell 2012, 148, 349-361. (15) Su, S.; Liu, Q.; Chen, J.; Chen, J.; Chen, F.; He, C.; Huang, D.; Wu, W.; Lin, L.; Huang, W.; Zhang, J.; Cui, X.; Zheng, F.; Li, H.; Yao, H.; Su, F.; Song, E. Cancer cell 2014, 25, 605-620. (16) Rokavec, M.; Öner, M. G.; Li, H.; Jackstadt, R.; Jiang, L.; Lodygin, D.; Kaller, M.; Horst, D.; Ziegler, P. K.; Schwitalla, S.; Slotta-Huspenina, J.; Bader, F. G.; Greten, F. R.; Hermeking, H. J. Clin. Invest. 2014, 124, 1853-1867. (17) Shi, Y.; Du, L.; Lin, L.; Wang, Y. Nat. Rev. Drug Discov. 2017, 16, 35–52. (18) Davis, F. M.; Stewart, T. A.; Thompson, E. W.; Monteith, G. R. Trends Pharmacol. Sci. 2014, 35, 479-488. (19) Shibue, T.; Wernberg, R. A. Nat. Rev. Clin. Oncol. 2017, 14, 611-629. (20) Marcucci, F.; Stassi, G.; De Maria, R. Nat. Rev. Drug Discov. 2016, 15, 311-325. (21) Pattabiraman, D. R.; Bierie, B.; Kober, K. I.; Thiru, P.; Krall, J. A.; Zill, C.; Reinhardt, F.; Tam, W. L.; Weinberg, R. A. Science 2016, 351, aad3680. (22) Holohan, C.; Schaeybroeck, S. V.; Longley, D. B.; Johnston, P. G. Nat. Rev. Cancer 2013, 13, 714-726. (23) Guptal, S.; Maitra, A. Cell 2016, 164, 840-842. (24) David, C. J.; Huang, Y.; Chen, M.; Su, J.; Zou, Y.; Bardeesy, N.; Iacobuzio-Donahue, C. A.; Massague, J. Cell 2016, 164, 1015-1030. (25) Carmona-Cuenca, I.; Roncero, C.; Sancho, P.; Caja, L.; Fausto, N.; Fernandez, M.; Fabregat, I. J. Hepatol. 2008, 49, 965-976. (26) Gal, A.; Sjoblom, T.; Fedorova, L.; Imreh, S.; Beug, H.; Moustakas, A. Oncogene 2008, 27, 1218-1230. (27) Byers, L.; Diao, L.; Wang, J.; Saintigny, P.; Girard, L.; Peyton, M.; Shen, L.; Fan, Y.; Giri, U.; Tumula, P. K.; Nilsson, M. B.; Gudikote, J.; Tran, H.; Cardnell, R. J.; Bearss, D. J.;

Warner, S. L.; Foulks, J. M.; Kanner, S. B.; Gandhi, V.; Krett, N.; Rosen, S. T.; Kim, E. S.; Herbst, R. S.; Blumenschein, G. R.;. Lee, J. J.; Lippman, S. M.; Ang, K. K.; Mills, G. B.; Hong, W. K.; Weinstein, J. N.; Wistuba, I. I.; Coombes, K. R.; Minna, J. D.; Heymach, J. V. Clin. Cancer Res. 2012, 19, 279-290. (28) Wong, I. Y.; Javaid, S.; Wong, E. A.; Perk, S.; Haber, D. A.; Toner, M.; Irimia, D. Nat. Mater. 2014, 13, 1063−1071. (29) Yu, Z.; Sun, Q.; Pan, W.; Li, N.; Tang, B. ACS Nano 2015, 9, 11064-11074. (30) Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C.; Mirkin, C. A.; Thaxton, C. S. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17104−17109. (31) Gao, W.; Ji, L.; Li, L.; Cui, G.; Xu, K.; Li, P.; Tang, B. Biomaterials 2012, 33, 3710-3718. (32) Yang, L.; Chen, Y.; Pan, W.; Wang, H.; Li, N.; Tang, B. Anal. Chem. 2017, 89, 6196-6201. (33) Li, N.; Chang, C.; Pan, W.; Tang, B. Angew. Chem., Int. Ed. 2012, 51, 7426-7430. (34) Luan, M.; Yu, L.; Pan, W.; Gao, X.; Wan, X.; Li, N.; Tang, B. Anal. Chem. 2017, 89, 10601-10607. (35) Luan, M.; Li, N.; Pan, W.; Yang, L.; Yu, Z.; Tang, B. Chem. Commun. 2017, 53, 356-359. (36) Pan, W.; Yang, H.; Li, N.; Tang, B. Chem. - Eur. J. 2015, 21, 6070-6073. (37) Tachibana, I.; Imoto, M.; Adjei, P. N.; Gores, G. J.; Subramaniam, M.; Spelsberg, T. C.; Urrutia, R. J. Clin. Invest. 1997, 99, 2365-2374. (38) Bachmeier, B. E.; Nerlich, A. G.; Iancu, C. M.; Cilli, M.; Schleicher, E.; Vené, R.; Dell'Eva, R.; Jochum, M.; Albini, A.; Pfeffer, U. Cell Physiol. Biochem. 2007, 19, 137-152. (39) Huang, T.; Chen, Z.; Fang, L. Oncol. Rep. 2013, 29, 117-124. (40) Gore, A. J.; Deitz, S. L.; Palam, L. R.; Craven, K. E.; Korc, M. J. Clin. Invest. 2014, 124, 338-352. (41) Yu, Z.; Pan, W.; Li, N.; Tang, B. Chem. Sci. 2016, 7, 4237– 4244.



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