GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High

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Biological and Medical Applications of Materials and Interfaces

GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High Affinity to #v#3 Integrin for Precise Early Tumor Identification Zhenwei Yuan, Lijuan Gui, Jinrong Zheng, Yisha Chen, Sisi Qu, Yuanzhi Shen, Fei Wang, Murat Er, Yue-Qing Gu, and Haiyan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09841 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High Affinity to αvβ3 Integrin for Precise Early Tumor Identification Zhenwei Yuan, Lijuan Gui, Jinrong Zheng, Yisha Chen, Sisi Qu, Yuanzhi Shen, Fei Wang, Murat Er, Yueqing Gu*, Haiyan Chen* Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China

*Author to whom correspondence should be addressed: Haiyan Chen, PhD Email: [email protected] Tel: +86-25-83271080 Fax: +86-25-83271046 Yueqing, Gu PhD Email: [email protected]

Keywords: near infrared florescence, responsive, glutathione monitoring, tumortargeting, early tumor diagnosis

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Abstract The development of tumor-associated, stimuli-driven, turn-on near-infrared (NIR) fluorophores required urgent attention because of their potential in selective and precise tumor diagnosis. Herein, we described a NIR fluorescent probe (CyA-cRGD) comprised of fluorescence reporting unit (a cyanine dye) linked with GSH responsive unit (nitroazo aryl ether group) and tumor-targeting unit (cRGD). The NIR fluorescence of CyA-cRGD with sensitive and selective response to GSH can act as a direct off-on signal reporter for the GSH monitoring. Notably, CyA-cRGD possesses improved biocompatibility compared with CyA, which is highly desirable for in vivo fluorescencetracking of cancer. Confocal fluorescence imaging confirmed the tumor-targeting capability and GSH detection ability of CyA-cRGD in tumor cells, normal cells, coincubated tumor / normal cells and the 3D multicellular tumor spheroid. Furthermore, it was validated that CyA-cRGD could detect tumor precisely in GSH and integrin αvβ3 high expressed tumor-bearing mouse models. Importantly, it was confirmed that CyAcRGD possessed high efficiency for early-stage tumor imaging in the mouse models with the tumor cells implanted within 72 h. This method provided significant advances toward more in-depth understanding and exploration of tumor imaging, which may potentially be applied for clinical early tumor diagnosis.

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Introduction Even with the improvement of medications towards cancer treatment, early and accurate cancer diagnosis is yet to be deciphered.1,

2

The numerous advances in

developing new imaging technologies including magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computerized tomography (SPECT) for cancer diagnosis have contributed to the continuous decline in the cancer mortality rate,3,

4

but they lack the efficacy of innocuous and targeting capability to

simultaneously facilitate the real-time delineation of diseased tissue while preserving vital tissues. Compared to other imaging techniques, the near infrared (NIR) fluorescence imaging is non-invasive, relatively inexpensive and simple to perform, which can be attributed to its molecular fluorescence as an exogenous contrast agent.5 To enhance deep tissue penetration and prevent interference associated with scattering and autofluorescence caused by biomolecules in tissues,6 truly groundbreaking NIR fluorophores such as small organic dyes,7 quantum dots (Q-dots)8 and upconversion nanoparticles (UCNPs)9 have been developed with current imaging system. Especially, advances in oncology, bioinformatics and proteomics have facilitated the development of novel fluorescence probes for tumor diagnosis that have decreased side effects and increased sensitivity, selectivity and biocompatibility.10-12 Efforts to identify cancer biomarkers that are found explicitly in tumor microenvironments and inside cancer cells have produced approaches for targeting delivery of fluorescence probes, which are essential methods for selective cancer imaging.13, 14 The diagnostic contrast agents are active targeting because their tethered tumor-targeting moiety can recognize the biomarkers with respect to enzyme,15 nonenzymatic protein16 and GSH17-21 expression, mRNAs,22 ROS stress levels23 and microenvironment acidities.24 Choi’s group,25 Low’s group,26 and other groups27-29 reported several fluorescent probes by connecting tumor-targeting groups to NIR fluorophores (such cyanine derivative,30 dicyanomethylene-4H-pyran31 and rhodol derivative32), which showed high signal-to-background ratio (SBR) associated with reduced nonspecific binding. However, this strategy would be limited when the tumor fails to express large quantities of the surface biomarkers, which significantly lowers the SBR.33,

34

Meanwhile, the molecular weight and size of the contrast domain and the

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excretion pathway have to be included in consideration of designing active-targeting fluorophores. Additionally, many activation strategies based on anomalies of cancer cells have also been exploited in designing the diagnostic fluorophores. The intrinsic fluorescence,35 ultrasonic wave,36 magnetic field37 or other visualizing signals38, 39 enhanced dramatically the following activation by particular internal stimulus (i.e., pH,40 concentration of metabolite,41 enzyme,42 redox potential in hypoxic cells,43 etc). These turn-on strategies are generally preferred over their turn off counterparts, owing to their lower background and high SBR in tumor diagnosis. Unfortunately, this can be a prolonged process with the nonspecific distribution of the molecules.25 And the in vivo images displayed a high background in the tissues surrounding the tumors.44 A noteworthy yet less researched goal is to achieve the cancer-targeting, stimuli-driven, turn-on diagnosis through an internal stimulus. In view of these significant challenges, we herein develop a novel NIR GSH-activated probe (CyA-cRGD) for early and accurate cancer diagnosis based on the high affinity to αvβ3 integrin. 3. Results and Discussion 3.1 Design and Synthesis A tumor-targeting turn-on NIR fluorescence probe, particularly suitable for early tumor diagnosis, was designed for in vivo diagnosis of the tumor in different stages. This probe was comprised of a fluorescence reporting unit (a cyanine dye), a tumor-targeting unit (cRGD), and a GSH responsive unit (nitroazo aryl ether group). The proposed off-on fluorescence mechanism of CyA-cRGD activated by GSH is depicted in Scheme 1. The ideal positions and shapes indicate that cRGD still maintained the high affinity to integrin αvβ3 after conjugation with CyA. The synthetic route diagram of CyA-cRGD is displayed in Scheme 2. The key intermediate compound (fluorescence report unit, cyanine dye) was synthesized according to the established procedures,45 and then conjugated with GSH responsive unit directly. The obtained moiety was linked with the tumor-targeting group through glutamic anhydride linker to provide a target compound CyA-cRGD with a satisfactory yield of 76.6 %. All the structures of intermediate products and CyA-cRGD were validated by 1H NMR,

13

C NMR and MALD-TOF-MS spectra (Figure S1-S19).

The nitroazo ether group was introduced as the GSH-responsive unit as well as the

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fluorescence quencher unit.46 The probe was expected to achieve significant fluorescence enhancement through a substitution reaction of the azo group by GSH and outstanding tumor-targeting capability through the affinity between cRGD moiety and integrin αvβ3.

Scheme 1. Proposed activation mechanism of the probe CyA-cRGD mediated by GSH.

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Scheme 2. The Synthesis route of CyA-cRGD.

3.2 GSH-Activable Optical Properties The GSH-induced substitution reaction influences the photophysical properties of CyA-cRGD. Owing to its donor-π-acceptor (D-π-A) structure, CyA-cRGD exhibited a broad absorption band at 782 nm with fairly weak emission at 802 nm. Its absorbance increased distinctly as the reaction time extended after addition of GSH (Figure 1A). Accordingly, the fluorescence spectra of CyA-cRGD displayed an evident red-shift (λem: 784 to 810 nm) of its fluorescence peak with remarkable fluorescence enhancement (~ 30 fold) upon the addition of GSH. The time-dependent fluorescent response of CyA-cRGD towards GSH (1 mM) stabilized at t=150 min (Figure 1B). Meanwhile, the inserted NIR fluorescence images in Figure 1B also indicated an apparent fluorescence difference of CyA-cRGD before and after (150 min) the addition of GSH. Furthermore, as shown in Figure 1C and Figure 1D, the absorbance and fluorescence intensity of CyA-cRGD gradually increased as GSH added, and eventually stabilized when GSH concentration reached 2.0 mM (Figure 1E). Notably, the peak fluorescent intensities at 810 nm showed a linear relationship with GSH concentrations in the range of 0 - 20 µM L-1 (Figure 1F), indicating that the fluorescent intensity of the CyA-cRGD in this range can be used to quantify GSH. From the linear range of the titration plot, the detection limit of GSH was determined to be as low as 0.26 µM, indicating a superior sensitivity in detecting GSH. The above results demonstrated that CyA-cRGD was an efficient turn-on NIR fluorescent probe for GSH detection.

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Figure 1. Time-dependent (A) absorption spectra and (B) fluorescence spectra of activable probe CyA-cRGD (10 µM) in the presence of GSH (1.0 mM) in HEPES/DMSO solution (99/1, 0.10 M, pH 7.4). Inset: corresponding fluorescence images of CyA-cRGD detected by NIR fluorescence imaging system; (C) Absorption spectra and (D) Fluorescence spectra of CyA-cRGD (10 µM, λex 760 nm) with the incremental addition of GSH concentration (0 - 10.0 mM) in HEPES buffer (0.10 M, pH 7.4); (E) The concentrations (0 - 10.0 mM) of GSH was plotted as the fluorescence intensity of CyA-cRGD changed; (F) The linear plot of the fluorescence intensity of CyA-cRGD as the concentrations of GSH ranged from 0 - 20 µM.

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NEM, as a scavenger for thiol,47 it can react with GSH but not with CyA-cRGD. The effect of NEM (10 mM) on the fluorescence of CyA-cRGD (10 µM) with and without the addition of GSH (10 mM) was investigated. There was no apparent fluorescent change in the control group (CyA-cRGD, CyA-cRGD + NEM). However, a significant NIR fluorescence was observed upon excitation at 760 nm after addition of GSH and exhibited apparent red-shift from 784 nm to 810 nm. Upon the further inclusion of 10 mM NEM, the enhanced fluorescence of CyA-cRGD + GSH solution quenched distinctly, accompanied by the apparent blue shift from 810 nm back to 784 nm (Figure 2A and 2B). The inserted images in Figure 2B exhibited the NIR fluorescence images of different samples, further indicated that NEM was an excellent inhibitor of GSH. Subsequently, various concentrations (0 - 10 mM) of NEM were utilized to validate the fluorescent response of CyA-cRGD towards GSH. In the presence of GSH (1.0 mM), the absorption band shifted to 770 nm with a 14 nm blue shift, accompanied by noticeable absorption reduction as the amount of NEM increased (Figure 2C). Meanwhile, the fluorescence peak decreased observably and was characterized by a blue shift from 810 nm to 784 nm (Figure 2D). The results substantiated that GSH caused the significant NIR fluorescence turn-on of CyA-cRGD with noticeable Stokes shift.

Figure 2. (A) Absorption spectra and (B) fluorescence spectra of CyA-cRGD, CyA-cRGD +

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NEM, CyA-cRGD + GSH, and CyA-cRGD + NEM + GSH (10 mM). Inset: corresponding NIR fluorescence images of CyA-cRGD detected by NIR fluorescence imaging system. (C) Absorption spectra and (D) fluorescence spectra of CyA-cRGD (10 µM) in the presence of NEM (0-10 mM) and GSH (10 mM) in HEPES buffer (0.10 M, pH 7.4).

The selectivity of CyA-cRGD towards GSH is one of the most important factors for the accuracy of GSH detection in a complicated biological environment. The interaction of CyA-cRGD with other analytes that engender biological interference such as amino acids and abundant metal ions was investigated to evaluate the potential application of CyA-cRGD in real biological systems. As described in Figure 3, upon exposure to various amino acids (GSH, Cys, Glu, Gly, Hcy, His, Phe, Ser, Val, Asp, Ala, Arg), only CyA-cRGD with the addition of GSH exhibited a significant fluorescence increase at λmax 810 nm. Besides, no substantial fluorescence enhancement of CyA-cRGD could be observed in the presence of different metal ions (Na+, Mg2+, Ca2+), further confirming the high selectivity of CyA-cRGD in a biological system. Actually, the potential interference of Cys and Hcy can be neglected due to their relatively low concentration in contrast to GSH with high physiological concentration in cytoplasm (1-15 mM).48, 49 Furthermore, the pH dependence of the emission spectra of CyA-cRGD with GSH was investigated (Figure S20). It showed a stable fluorescence intensity in a broad pH range (6.5 to 10.5), which is beneficial for GSH tracking in living systems. Also, the response of CyA-cRGD to GSH in different solvent conditions indicated that HEPES buffer containing 1 % DMSO could be selected as the optimal system for the research of CyA-cRGD (Figure S21). Therefore, the results demonstrated that CyA-cRGD could be served as a highly selective fluorescent probe for GSH. Further, the stability of CyA-cRGD in blood after post injection was verified. As shown in the Figure S22, in the presence of serum, the fluorescence intensity of CyA-cRGD has faint enhancement, it indicated that CyA-cRGD is relatively stable enough to be used as an effective targeting and responsive fluorescence probe for tumor imaging.

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Figure 3. (A) Relative fluorescence intensity ratios of CyA-cRGD (10 µM) with the addition of 10 mM various amino acids (GSH, Cys, Glu, Gly, His, Phe, Ser, Val, Asp, Ala, Arg, Hcy) and 10 mM metal ions (Na+, Mg2+, Ca2+). Insert: Fluorescence spectra of CyA-cRGD (10 µM) to various amino acids and metal ions (10 mM). F and F0 represent the fluorescence intensity of CyA-cRGD with or without the addition of GSH, respectively; (B) Corresponding fluorescence images of CyA-cRGD in the presence of different amino acids and metal ions detected by NIR fluorescence imaging system.

The distinctly different spectroscopic response of CyA-cRGD to GSH could be attributed to the cleavage of the electron-withdrawing nitroazo aryl ether group. This also indicated the successful conjugation of the cyanine dye and GSH responsive moiety (nitroazo aryl ether group). Notably, the fluorescence of CyA-cRGD fell into the NIR region (700-900 nm), particularly preferable for in vivo bioimaging because of the deep penetration ability and low background autofluorescence. Furthermore, the significant fluorescence enhancement resulting from activation by GSH is desirable for high quality optical imaging because it enhances the signal to noise ratio dramatically. 50, 51 3.3 Reaction Mechanism Study To validate that photoinduced electron transfer (PeT) from the cyanine dye to nitroazo ether group would lead to fluorescence “non-emission” before the substitution by the azo group of GSH, the lowest-energy absorption and singlet emission of CyA-cRGD and CySG were calculated computationally using the B3LYP 6-31g(d) level of density functional theory (DFT). The natural transition orbitals (NTOs) depicted in Figure 4 suggested that nitroazo ether group raised both the HOMO and LUMO of the cyanine dye segment, enhancing the feasibility of PeT from the cyanine dye to nitroazo ether group

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while preventing PeT from the HOMO of Cy-SG. The electron cloud density of the cyanine dye core in CyA-cRGD decreased due to PeT, rendering a dark compound. Whereas, the electron cloud density of Cy-SG increased, which was contributed by the intramolecular charge transfer and the conjugated p-π system between S atom and cyanine dye, thus Cy-SG could emit strong NIR fluorescence. The energy gaps (HOMO−LUMO) of CyA-cRGD and Cy-SG were calculated as 0.07582 eV and 0.070 eV respectively, validating the fluorescence red-shift of Cy-SG compared to CyA-cRGD. Calculations based on DFT were utterly consistent with the experimental results mentioned above. Moreover, to demonstrate Cy-SG is the product of GSH-catalyzed CyA-cRGD, the MALD-Tof/Tof-MS spectrometric analysis was performed and proved that the response of CyA-cRGD to GSH was related to the deduced substitution mechanism (Figure S23). The observation of a peak at m/z = 926.4373 in the MALDTof/Tof-MS spectrum was corresponding to Cy-SG ([M+] 926.4368) (Figure S23). The result confirmed that the nitroazo of CyA-cRGD was substituted with a thiol group, which contributed to the marked fluorescence enhancement of the probe after the inclusion of GSH.

Figure 4. The frontier molecular orbitals (MOs) of (A) CyA-cRGD and (B) Cy-SG involved in the vertical excitation (the left columns) and emission (right columns). The vertical excitation and emission related calculations were performed computationally using the B3LYP 6-31g(d) level of theory. The electron distribution at HOMO and LUMO is shown in red and green color. The

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dashed line represents the internal conversion.

3.4 Cell affinity evaluation of CyA-cRGD and visualization of GSH in Cells

Figure 5. Cytotoxicity of CyA and CyA-cRGD in (A) L02 cells, (B) MCF-7 cancer cells, (C) U87MG cancer cells and (D) A549 cancer cells.

To evaluate the cell affinity of CyA-cRGD and its potential for visualization of GSH in biological systems, first, the biocompatibility of the probe was investigated. The cytotoxicity of CyA and CyA-cRGD against a large panel of normal cell lines and human tumor cell lines were determined using conventional MTT assays (Figure 5). CyA-cRGD showed significant low biotoxicity against cell lines including L02, MCF-7, U87MG, and A549. Compared with CyA-cRGD, CyA showed higher cytotoxicity against cells, especially the viability of A549 cells were only 56.3 % at high incubation concentration (20 µM L-1). Obviously, the results indicated CyA-cRGD possessed greater biocompatibility than CyA due to the contribution from the endogenesis and hydrophilicity of cRGD, whereas CyA was a typical cyanine dye and characterized by hydrophobicity in the biological system, resulting in higher cytotoxicity. Undoubtedly,

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CyA-cRGD released more cRGD in cancer cells rather than normal cells because of the much higher concentration of intracellular GSH in cancer cells,52, 53 resulting in lower cell viability in cancer cells than in normal cells. The favorable biocompatibility lent credence to the applicability of CyA-cRGD as an effective bioprobe.

Figure 6. Schematic illustration of the performance and the fluorescence recovery process of CyA-cRGD in vivo.

As we know, the GSH concentration of cancerous cells always higher than normal cells, and concentration range from 1 to 10 mM in cancer cells.54-56 Thus, after confirming the sensitivity, selectivity, and biocompatibility of the probe, the potential of CyA-cRGD for the intracellular GSH monitoring and cell affinity evaluation were further explored on GSH and integrin αvβ3 over-expressed cells (MDA-MB-231 cells and A549 cells), as well as GSH and integrin αvβ3 low-expressed cells (L02). The performance and the fluorescence recovery process of CyA-cRGD in cells are first illustrated in Figure 6.

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MDA-MB-231 cells displayed intense red luminescence 30 min after incubation with CyA-cRGD consistently, where the green fluorescence in mitochondria was visualized by MitoTracker Green staining (Figure 7A). Additionally, more prolonged incubation of the cells with the probe showed an increased fluorescence of red channel accompanied by a dramatic color change of merged channel from green to yellow, revealing a substantial overlap of the fluorescence signal from Cy-cRGD and that from Mito Tracker Green. The semi-quantitative analysis of fluorescence intensity as the incubation time (0 h to 4 h) extended is shown in Figure 7B. These results constituted the first direct visualization of GSH activity localized explicitly in mitochondria rather than other organelles, which was in good agreement with the GSH-elicited spectroscopic response of CyA-cRGD. As depicted in Figure 7C and Figure 7D, the fluorescence from CyA-cRGD (red) was observed in the mitochondria (green) after 2 h of incubation at 37 °C, indicating that CyA-cRGD was quickly taken up by MDA-MB-231 cells and decomposed into Cy-SG by the intracellular GSH. By adding an extra 1.0 mM NEM (a known inhibitor for GSH) to inhibit the expression level of GSH, a dramatic decrease of fluorescence was observed in the cells incubated with CyA-cRGD. In contrast, the fluorescence of CyA-cRGD showed a visible recovery provided that GSH was continuously added into the NEM inhibited MDA-MB-231 cells, which further validated the selective and activable fluorescence response of CyA-cRGD to GSH. Furthermore, the cell affinity of CyAcRGD contributed by the targeting moiety (cRGD) was also investigated simultaneously at the cell level. The cells pretreated with 1.0 mM cRGD showed significantly weaker fluorescence (red) than those cultivated with only CyA-cRGD, which validated that less CyA-cRGD was uptaken by MDA-MB-231 cells because of the saturation of the integrin αvβ3 acceptor on the cell membrane after the addition of cRGD. Excitedly, the fluorescence signal within the mitochondria after the cells were incubated with additional NEM and cRGD synchronously indicated a further attenuation of the red fluorescence, which demonstrated that the probe responded to GSH sensitively and bound to integrin αvβ3 selectively. The semi-quantitative analysis of the fluorescence intensity in different groups is displayed in Figure 7D. In addition, the scatter plot and correlation plot of Mito-Tracker Green and CyA-cRGD in MDA-MB-231 cell fluorescence images were exhibited (Figure 7E). The fluorescent signal of linear regions of interest (ROI) showed a

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tendency to synchronize with a Pearson’s colocalization coefficient of 0.9255. As shown in Figure S24, the fluorescence of CyA-cRGD in A549 cells also displayed the timedependent enhancement, as well as GSH activable fluorescence response and integrin αvβ3 acceptor-mediated targeting capability. The Pearson’s colocalization coefficient of red and green fluorescence in A549 cells were 0.9346. The results further suggested that CyA-cRGD could specifically accumulate in mitochondria, indicating that this probe is capable of monitoring GSH located in mitochondria. Meanwhile, the contribution of the tumor-targeting moiety cRGD for the affinity to the tumor cells was comprehensively validated.

Figure 7. LCFM fluorescence images of CyA-cRGD in MDA-MB-231 cells. (A) Time

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dependent cellular imaging of CyA-cRGD (2.0 µM) on MDA-MB-231 cell lines monitored by LCFM; (B) Semi-quantification of the fluorescence intensity of CyA-cRGD in MDA-MB-231 cells at different incubation time; (C) LCFM fluorescence images of MDA-MB-231 cells in the presence of CyA-cRGD (2.0 µM) only, CyA-cRGD (2.0 µM) + NEM (1 mM), CyA-cRGD (2.0 µM) + NEM (1 mM) + GSH (1 mM), CyA-cRGD (2.0 µM) + cRGD (1 mM) and CyA-cRGD + NEM (1 mM) + cRGD (1 mM); (D) Semi-quantification of the fluorescence intensity of CyAcRGD in MDA-MB-231 cells in different conditions; (E) Linear profile and scatter plot were used to characterize the overlap degree of red fluorescence from CyA-cRGD and green fluorescence from Mito Tracker Green. Error Bar: 20 µm.

Next, the potential of CyA-cRGD for the intracellular GSH imaging and its targeting capability were evaluated by using GSH and integrin αvβ3 low-expressed normal cells (L02). As indicated in Figure 8A and 8B, the time-dependent fluorescence enhancement was not as evident in L02 cells as in MDA-MB-231 or A549 cells. Furthermore, Figure 8C and Figure 8D displayed that the red fluorescence emitted from only CyA-cRGD incubated L02 cells was as weak as NEM inhibited or cRGD saturated L02 cells. Although the red fluorescence decreased dramatically in L02 cells compared to MDAMB-231 cells, it still showed substantial overlap with the green fluorescence from Mito Tracker Green with a Pearson’s colocalization coefficient of 0.94. Meanwhile, the affinity of CyA-cRGD to different types of cells was compared in Figure S25. All these results proved that the fluorescence intensity of the probe CyA-cRGD in cells was correlated with the expression level of GSH, and its cell affinity was related to the level of integrin αvβ3 overexpressed on cells. Further, we compared the cell affinity of CyA-cRGD and CyA that GSH responsive cyanine dye linked without cRGD. The potential of CyA-cRGD and CyA for the cell affinity evaluation was explored in GSH and integrin αvβ3 over-expressed cells (MDAMB-231 cells and A549 cells), as well as GSH and integrin αvβ3 low-expressed cells (L02). The performance of CyA and CyA-cRGD in cells are first illustrated in Figure S26. MDA-MB-231 cells and A549 cells displayed intense red fluorescence 2 h after incubation with CyA-cRGD consistently, where the green fluorescence in mitochondria was visualized by Mito Tracker Green staining. However, MDA-MB-231 cells and A549 cells displayed weak red fluorescence 2 h after incubation with CyA. Additionally, L02

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cells displayed weak red fluorescence with both CyA and CyA-cRGD. This result further demonstrated the superiority of our strategy designed probe.

Figure 8. LCFM fluorescence images of CyA-cRGD in L02 cells. (A) Time dependent cellular imaging of CyA-cRGD (2.0 µM) on L02 cell lines monitored by LCFM; (B) Semi-quantification of the fluorescence intensity of CyA-cRGD in L02 cells at different incubation time; (C) LCFM fluorescence images of L02 cells in the presence of CyA-cRGD (2.0 µM) only, CyA-cRGD (2.0 µM) + NEM (1 mM), CyA-cRGD (2.0 µM) + NEM (1 mM) + GSH (1 mM), CyA-cRGD (2.0

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µM) + cRGD (1 mM) and CyA-cRGD + NEM (1 mM) + cRGD (1 mM); (D) Semi-quantification of the fluorescence intensity of CyA-cRGD in L02 cells in different conditions; (E) Linear profile and scatter plot were used to characterize the overlap degree of red fluorescence from CyA-cRGD and green fluorescence from Mito Tracker Green. Error Bar: 20 µm.

The investigation of the performance of the probes in the cancer cells and normal cells co-cultured system is a practical approach to evaluate their specificity and targeting capability.57,

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Inspired by this strategy, U87 and L02 cells were selected to be co-

cultured in one confocal dish to examine the potential of CyA-cRGD for tumor-targeting imaging and GSH monitoring in tumor cells (Figure 9 and Figure S27). After co-cultured U87 and L02 cells were incubated with CyA-cRGD for 1 h, strong red fluorescence was observed in the cytoplasm of U87 cells, whereas extremely weak fluorescent signal was found in L02 cells (Figure 9A and Figure 9B), demonstrating the specificity and reliability of CyA-cRGD for cancer cells in the complicated biological system. The green fluorescence emitted from mitochondria (Figure 9C) and the yellow fluorescence shown in the merged image (Figure 9D) corroborated the selective accumulation of the probe in mitochondria of U87 cells. Figure 9E exhibited the reconstructed 3D fluorescence image for this co-cultured cell system. Semi-quantification of the fluorescence intensity of CyAcRGD in cells further supported that CyA-cRGD entered U87 cells cytoplasm more efficiently than L02 cells. (Figure 9F) The different fluorescence intensity shown in U87 cells and L02 cells also accorded with different GSH levels in these two cell lines. Therefore, CyA-cRGD provided a promising prospect for monitoring intracellular GSH levels and distinguishing cancer cells from normal cells. It is essential to evaluate the selectivity of the probe in complicated biological system containing both cancer and normal cells. Unlike traditional cell culture method, the cocultured cells system provides a more reliable way to mimic the real biological environment. Ma et al. utilized co-cultured tumor and normal cells to assess the molecular beacon named AuNP-MBs and demonstrated the specificity and reliability of AuNP-MBs by this methodology.59 Herein, the high selectivity of CyA-cRGD shown in the co-cultured U87 and L02 cell system laid a potent foundation for the further investigation of this probe in vivo.

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Figure 9. (A) Differential interference contrast image of U87 cells and L02 cells. (B) The fluorescence image of U87 cells and L02 cells incubated with CyA-cRGD (100 µL, 1 µM) collected at NIR channel (780 ± 30 nm, λex = 690 nm). (C) The green fluorescence (MitoTracker Green) image of mitochondria in U87 cells and L02 cells. (D) The Merged fluorescence image U87 cells and L02 cells; (E) The reconstructed 3D fluorescence image for co-cultured U87 cells and L02 cells. (F) Semi-quantification of the red and green fluorescence intensity in U87 cells and L02 cells. Error Bar: 20 µm.

3.5 In vitro penetration of CyA-cRGD into the 3D U87 tumor spheroid To further verify the tumor penetration capability of CyA-cRGD, the tumor spheroids, 3D U87 cell culture system was introduced to monitor the penetration of the probe in a vivo-mimic tumor (Figure 10). After the tumor spheroid incubated with CyA-cRGD for 6 h, the bright red fluorescence spread in the majority of regions, even 60 µm from the surface towards the middle. It was determined to penetrate approximately 33 % of the tumor spheroid which has a radius of about 180 mm. Hochest 33342 was used to localize the tumor cell nucleus, and the merged images demonstrated the red fluorescence from the probe overlapped with the blue fluorescence from cell nuclei substantially. Therefore CyA-cRGD can penetrate into the tumor spheroid in a large extent. Moreover, no

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remarkable change was observed in the morphology of tumor spheroids incubated with the probe, which further validated the biocompatibility of CyA-cRGD. Development of physiologically-related in vitro tumor models serves as an important role in tumor research.60 2D monolayer cell culture is commonly used to evaluate the efficacy of biomolecules, antitumor drugs, and probes. However, this simplified model is deficient of cell–cell and cell–matrix interactions and could not mimic the in vivo complicated biological environment perfectly, thus the correlation between in vitro culture, mouse in vivo results, and clinical responses was very poor. Sutherland et al. were first to propose multicellular tumor spheroids (MTS) as a 3D model of small solid tumors.61 The 3D spheroids introduced recently were compact, stable and showed oxygen levels, and nutrition supplies from the periphery toward the spheroid’s center.62, 63 The physiological characteristics of the 3D spheroid closely resembled avascular tumor nodules, micro-metastases, and inter-vascular regions of large solid tumors.64 Therefore, it is a convictive tactic to approve the efficient delivery of CyA-cRGD from the exterior to the interior of the solid tumor by the 3D U87 tumor spheroid.

Figure 10. In vitro penetration of CyA-cRGD into the 3D multicellular U87MG tumor spheroids after incubation with CyA-cRGD for 6 h and Hochest for 0.5 h. (A) The schematic illustration of the construction of the 3D spheroids model; (B) Z-stack images using LSCM were obtained from the top to the equatorial plane of the tumor spheroid in 15 µm thickness; (C) The 3D reconstructed fluorescence image was obtained from the top to the equatorial plane of the tumor spheroid; (D) Semi-quantitative analysis of fluorescence intensity. Error Bar: 100 µm.

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3.6 Western blot analysis CRGD peptides are small molecules that can bind to integrin αvβ3 with high affinity and can inhibit the αvβ3-mediated signal pathways competitively. FAK protein and ERK1/2 protein are two essential signal molecules that can be activated by integrin αvβ3 mediation and also phosphorylation of these two proteins can be inhibited by cRGD.65 Herein, the enzymatic assays were utilized to prove that CyA-cRGD underwent the same underlying molecular mechanisms with cRGD after binding to integrin αvβ3. It is well-known that integrin αvβ3 directly acts on the FAK protein and impacts the downstream Erk1/2 signaling pathways.66,

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Accordingly, Western blot analysis showed that CyA-cRGD

decreased the level of FAK, p-FAK, Erk1/2 and p-Erk1/2 significantly (Figure 11), implying that the probe possessed a high affinity with integrin αvβ3 due to the contribution of cRGD. In comparison with cRGD, the lower integrin αvβ3 affinity of CyA-cRGD can be attributed to stereo-hindrance of its larger molecular structure, whereas the binding sites were not blocked and only the binding efficiency was influenced in a certain extent. These results confirmed that CyA-cRGD, with the binding of integrin αvβ3, was functionally equivalent to cRGD and acted on same signaling pathways.

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Figure 11. (A) The schematic illustration of the integrin receptor-associated signal pathway; (B) Representative FAK, p-FAK, Erk1/2, p-Erk1/2, and GAPDH protein expression determined by Western blot analysis; (C) Semi-quantitative analysis of the Western blot result.

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3.7 Synchronous in vivo tumor-targeting and GSH-responsive imaging in different tumor-bearing mice model The promising results in living cell imaging inspired us to further explore the feasibility of CyA-cRGD as an in vivo effective NIR fluorescence probe for tumortargeting and GSH monitoring. The biocompatibility of CyA-cRGD as a fluorescent probe for animal bioimaging in vivo was investigated first by assessing its biotoxicity (Figure S28). The major organs (heart, liver, spleen, lung, and kidney) were excised from normal mice at 7 days post-injection of the probe, and histological analysis was performed afterwards. In comparison with the controlled group, there were no signs of degenerative and necrotic changes of the parenchymatous cells, interstitial proliferation or inflammatory reaction in the major organs of CyA-cRGD injected group, indicating the favorable biocompatibility of the probe in animal level. The NIR fluorescence images of CyA-cRGD or CyA-cRGD + NEM in normal mice were collected to kinetically observe the change in the fluorescence intensity of CyAcRGD before and after injection into the mouse. Utilizing NIR fluorescence to strengthen the tissue penetration depth and the contrast ratio, CyA-cRGD can overcome the interference from the biological auto-fluorescence. As shown in Figure 12A, the intense fluorescence signal appeared in liver only 10 min post-injection of the probe, and it lasted till 24 h post-injection. Also, another group of mice was pretreated with 100 µM NEM for 30 min to inhibit the concentration of GSH in mice. Notably, the clear fluorescence signal at liver did not appear till 4 h post-injection of the probe, and it was maintained till 24 h post-injection (Figure 12B). The fluorescence images of isolated tissues at the corresponding time points and the semi-quantitative analysis further indicated the different fluorescence changes in these two groups, especially the difference in the GSH high expressed liver (Figure 12C and Figure 12D). Based on the fluorescence intensity in different organs (heart, liver, spleen, lung, kidney, and intestine), the heat map was established to reveal the influence of NEM on the distribution of the probe. As shown in Figure 12E, the spatial and temporal distribution of CyA-cRGD generally exhibits different profiles for the two groups (CyA-cRGD, NEM + CyA-cRGD). Due to the inhibition of NEM, the GSH expression level in liver was low at the first 2 h of in vivo imaging experiments. Therefore, the NEM inhibition group demonstrated extremely weak

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fluorescence in the first 2 h when compared with the only CyA-cRGD injected group. After 2 h, the inhibition effect eliminated gradually; thus, the enhanced fluorescence signal was shown in the liver of NEM treated group. Furthermore, the active fluorescence demonstrated in the intestine can be attributed to the enterohepatic circulation and metabolism of the probe, whereas other tissues all displayed completely weak fluorescence. All the results validated that the fluorescence of CyA-cRGD was responsive to GSH selectively so that the GSH expression level determined intensity.

Figure 12. Dynamic distribution of (A) CyA-cRGD, (B) NEM + CyA-cRGD in normal mice monitored by NIR fluorescence imaging system. The ex-vivo fluorescence images of isolated organs (heart, liver, spleen, lung, kidney, and intestine) from the normal mice at 0.5 h, 4 h, 8 h and 24 h post-injection of (C) CyA-cRGD, (D) NEM + CyA-cRGD; (E) Heat map of the spatial

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and temporal changes of the fluorescence intensity of CyA-cRGD and NEM + Cy-cRGD in main organs collected for different time intervals. (*P < 0.05).

CyA-cRGD was designed as an efficient probe that cannot only respond to GSH but also target tumor specifically. The in vitro validation laid a firm foundation for the further in vivo investigation. Herein, integrin αvβ3 over-expressed (MDA-MB-231, U87, A549 and H22) tumor-bearing mouse models, as well as integrin αvβ3 low expressed (MCF-7) tumor-bearing mouse models were all introduced to evaluate the suitability of CyAcRGD for in vivo tumor-targeting and GSH-responsive imaging synchronously. As shown in Figure 13A, the fluorescence signal initially spread over MDA-MB-231 tumor-bearing mouse body within 10 min of post-injection of CyA-cRGD. At 1 h post-injection of the probe, a distinct fluorescence signal was displayed in the tumor, revealing the active and specific tumor-targeting ability. The fluorescence signal reached peak intensity at 8 h and maintained the high contrast till 24 h. For NEM + CyA-cRGD injected group, no visible fluorescence was observed in the liver before 2 h of post-injection, whereas the prominent fluorescence started to appear in tumor at 1 h post-injection. The fluorescence signal gradually reached peak intensity at 12 h and maintained the high contrast ratio till 48 h (Figure 13B). Similar to the fluorescence images of NEM + CyA-cRGD injected normal mice, the weak fluorescence at the first 2 h was due to NEM inhibition and the recovery of GSH expression level led to the enhancement of the fluorescence intensity in liver after 2 h of post-injection. However, there was no apparent fluorescence discrepancy in tumor between CyA-cRGD injected group and NEM inhibition group. NEM is a commonly used GSH inhibitor, which can reach liver easily by metabolism but cannot reach tumor substantially due to the lack of positive targeting capability. Accordingly, the GSH expression level in liver was inhibited by NEM drastically but not in the tumor, resulting in the weak fluorescence production in the liver. In contrast, the tumor exhibited strong fluorescence, which can be attributed to the negligible influence from NEM and the high expression of integrin αvβ3 in the MDA-MB-231 tumor. To further validate the tumor-targeting effect of targeting moiety (cRGD) of the probe, MDA-MB-231 tumor-bearing mice were pretreated with additional cRGD, and NEM and the in vivo fluorescence imaging was performed. CyA-cRGD presented a significantly

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robust capability of tumor targeting in MDA-MB-231tumor-bearing mice. However, this specific tumor-targeting effect was almost entirely inhibited by the addition of cRGD and NEM from the initial 10 min to 48 h, as shown in Figure 13C. It was already proved that NEM did not influence the fluorescence signal appeared in the tumor; thus, reduction in the fluorescence was ascribed to the saturation of integrin αvβ3 by cRGD, leading to the dramatic reduction in fluorescence in tumor. Additionally, the influence of NEM on the fluorescence appeared in liver and the contrast ratio of tumor to normal tissue (T/N) in the three groups were further assessed and confirmed by a quantitative ROI analysis (Figure 13D and Figure 13E). Moreover, the synchronous in vivo tumor-targeting and GSH-responsive imaging by CyA-cRGD were also verified on other three (A549, U87 and H22) tumor-bearing mouse models with GSH and integrin αvβ3 high expression (Figure S29 to Figure S31).

Figure 13. In vivo fluorescence images of (A) CyA-cRGD, (B) NEM + CyA-cRGD and (C) NEM + cRGD + CyA-cRGD in MDA-MB-231 tumor-bearing mice model within 48 h; (D) Relative fluorescence intensity of belly in different mice group at a different post-injection time. (E) Tumor-to-normal tissue (T/N) ratios of the probe in tumor-bearing mice at a different post-

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injection time.

Although the expected tumor-targeting and GSH monitoring capability of CyA-cRGD were observed and verified in GSH and integrin αvβ3 high expressed tumor-bearing mouse models, its high selectivity, and specificity were further evaluated on GSH overexpressed, and integrin αvβ3 low expressed (MCF-7) tumor-bearing mouse model (Figure 14). Similarly, the strong fluorescence appeared immediately in liver and intestine rather than other organs (10 min) after the probe was intravenously injected and sustained up to 24 h. Meanwhile, NEM injected group demonstrated weak fluorescence initially and exhibited delayed strong fluorescence signal at 2 h after injection of CyA-cRGD, which was caused by the inhibition of GSH expression in liver upon the injection of NEM and the gradual removal of inhibition effect as time extended. In contrast, no notably visible fluorescence signal was shown in the tumors of all the three groups (CyA-cRGD, NEM + CyA-cRGD, NEM + cRGDCyA-cRGD, Figure 14A-C), which was corresponding to the low-expression of integrin αvβ3 in MCF-7 tumors, further indicating the selective and specific recognition capability of CyA-cRGD. Furthermore, the in vivo fluorescence images were assessed by ROI analysis (Figure 14D and Figure 14E). These data suggested that CyA-cRGD was sensitively responsive to GSH in vivo, as well as showed an enhanced tumor-targeting effect on integrin αvβ3 high-expressed tumors, which proved to be a promising NIR fluorescence probe with high sensitivity and selectivity for clinical applications.

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Figure 14. In vivo fluorescence images of (A) CyA-cRGD, (B) NEM + CyA-cRGD and (C) NEM + cRGD + CyA-cRGD in MCF-7 tumor-bearing mice models within 48 h; (D) Relative fluorescence intensity of belly in different mice group at a different post-injection time. (E) Tumor-to-normal tissue (T/N) ratios of the probe in tumor-bearing mice at a different postinjection time.

Since the in vitro and in vivo investigations confirmed the outstanding properties of CyA-cRGD for effective tumor-targeting and GSH-responsive imaging, the research on the efficiency of this probe for diagnosis of the tumor in the very early stage was further carried out. The schematic illustration of the construction of the initial (A549 and H22) tumor-bearing mouse models are shown in Figure 15A. CyA-cRGD was injected into the mice through tail vein upon 48 h, 72 h, 96 h, 120 h or 144 h after the tumor cells were injected into the axillary fossa of the mice respectively and the NIR fluorescence images were collected at 8 h post-injection of CyA-cRGD. As shown in Figure 15B and Figure

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15C, the clear fluorescence signal started to be detected in the implantation position at 72 h post-injection of the tumor cells. An increase in luminescence intensity was observed at implantation site the as the tumor cell implantation time extended from 96 h to 144 h. Meanwhile, the liver of the two tumor-bearing mice (A549 and H22) all displayed obvious fluorescence signal, which can be attributed to GSH-mediated fluorescence response of CyA-cRGD. Also, the ROI fluorescence analysis indicated the evident increase of tumor to normal tissue ratio (Figure 15E and Figure 15G). To further investigate the inner structure of the implantation position, the upper part of the mice body was dissected at 72 h post-injection of the tumor cells. As shown in Figure 15 D and Figure 15F, the tumor nodules and tumor angiogenesis were clearly observed in the implantation parts, indicating the apparently different inner structure compared to normal tissues. Meanwhile，the in vivo fluorescence images of CyA in the above two tumorbearing mouse models were also collected (Figure S32). No apparent fluorescence signals were observed from CyA injected tumor-bearing mice, even after the 6th day of the tumor cells were implanted into the mice, which further suggested the high sensitivity and efficiency of CyA-cRGD for early-stage tumor diagnosis.

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Figure 15. (A) The schematic illustration of the construction of the early-stage tumor-bearing mouse models; (B) CyA-cRGD was injected into the mice through tail vein upon 48 h, 72 h, 96 h, 120 h or 144 h after the A549 tumor cells were injected into the axillary fossa of the mice

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respectively and the NIR fluorescence images were collected at 8 h post-injection of CyA-cRGD; (C) The dissected photo of the mouse at 72 h after A549 tumor cells were implanted; (D)The change of A549 tumor to normal tissue ratios at different time intervals (48 h, 72 h, 96 h, 120 h or 144 h). (E) CyA-cRGD was injected into the mice through tail vein upon 48 h, 72 h, 96 h, 120 h or 144 h after the H22 tumor cells were injected into the axillary fossa of the mice respectively and the NIR fluorescence images were collected at 8 h post-injection of CyA-cRGD; (F) The dissected photo of the mouse at 72 h after H22 tumor cells were implanted; (G) The change of H22 tumor to normal tissue ratios at different time intervals (48 h, 72 h, 96 h, 120 h or 144 h).

According to the TNM Staging System,68 one of the most commonly used staging systems, which was developed and is maintained by the American Joint Committee on Cancer (AJCC) and adopted by the Union for International Cancer Control (UICC), the early tumor-bearing mouse models constructed (48 h, 72 h and 96 h) in this study were belonged to TX, NX, MO at different categories. CyA-cRGD was proved to be responsive to the cancer-associated stimuli (GSH) and could selectively accumulate in tumors (even in the early stage) but not in normal tissues both in vitro and in vivo. In order to enhance the efficiency of diagnosis, the active targeting strategy based on the conjugating ligands69 (e.g., cRGD, hyaluronic acid, transferrin) capable of interacting with specific receptors70 (e.g., integrin αvβ3, HAase, transferrin receptor) overexpressed on target cells were utilized to enhance tumor localizing. Meanwhile, those visualizing signals turned on upon recognition of the targeted biomolecules (e.g., GSH, Cathepsin B, Caspase-3, matrix metalloproteinases) further improved the signal contrast ratio of cancer to normal tissues. Most of existing turn-on fluorescent probes lack tumor-targeting capability, or the tumor-targeting fluorescent probes are not capable of providing detailed information about the tumor microenvironment.71 The synergetic contribution of GSHdriven activable fluorescence enhancement and ligand (cRGD)-acceptor (integrin αvβ3) mediated tumor-targeting capability facilitated CyA-cRGD to reveal outstanding performance on accurate and sensitive diagnosis of the tumor especially in the earl stage. Consequently, stimuli responsive turn-on NIR fluorescence probes with tumor-targeting ability will be able to provide an effective approach for precise, sensitive and early tumor diagnosis.

Conclusion

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In summary, we have enriched the principle and strategy for designing a probe for precise tumor detection and early tumor diagnosis. Experimental and theoretical calculation studies demonstrated the critical factors for designing a suitable probe, not only involving the charge transfer process induced by the electro-withdrawing group and GSH responsive fluorescence enhancement, but also introducing the tumor-targeting moiety to bind specifically to protein receptors that are highly expressed on tumors. On the basis of the principle, the successfully synthesized probe (CyA-cRGD) displayed high sensitivity, and excellent selectivity, with a fluorescence turn-on response to GSH and high affinity to integrin αvβ3. Kinetic optical studies and other experiments supported the proposed GSH catalytic reaction mechanism. Particularly, CyA-cRGD demonstrated improved biocompatibility compared with CyA due to the contribution of the tumortargeting moiety (cRGD). Confocal fluorescence imaging of tumor cells (GSH and integrin αvβ3 high expressed) and normal cells (GSH and integrin αvβ3 low expressed) confirmed the tumor cell affinity and GSH detection ability of CyA-cRGD at the cellular level and in the 3D multicellular tumor. Moreover, CyA-cRGD can detect tumor precisely in tumor-bearing mouse models with overexpressed GSH and integrin αvβ3, showing rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. More importantly, CyA-cRGD was proved to possess high efficiency for early-stage tumor diagnosis in the moue models with the tumor cells implanted within 72 h. CyA-cRGD not only can be used to precisely discern the tumors with high expression of GSH and integrin αvβ3 from the normal tissues but can also distinguish tumors in the very early stage from normal tissues in vivo, further highlighting the potential diagnostic application of CyA-cRGD. Our successful method provided significant advances toward the exploration of novel probes for highly sensitive in vivo GSH imaging studies and early tumor diagnosis. Also, we believe that in the coming years many conceptually new approaches in the design of cancer-targeting, stimuli-driven, turn-on fluorophores based on novel activation mechanisms will be devised, which will not only enable their use in cancer diagnostic applications, but also extend their use for in vivo image-guided cancer therapy.

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ASSOCIATED CONTENT Supporting Information Experimental section, 1H-NMR,

13

C-NMR and MALD-TOF-MS spectra of all the

structures, effect of pH and solvent on the absorbance and fluorescence intensity, the reaction mechanism of CyA-cRGD with GSH, LCFM fluorescence images of CyAcRGD in A549 cells, biotoxicity of CyA-cRGD on normal mouse model, in vivo fluorescence imaging in early tumor-bearing mice of CyA. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest.

Acknowledgement The authors are grateful to the Natural Science Foundation Committee of China (NSFC 81671803), a project supported by the National Key Research and Development Program (Grant No. 2017YFC0107700), the Outstanding Youth Foundation of Jiangsu Province (GX20171114003), the Postgraduate Research & Practice Innovation Program of Jiangsu Province and funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for their financial support.

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(4) Ferlay, J.; Soerjomataram, I.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D. M.; Forman, D.; Bray, F. Cancer Incidence and Mortality Worldwide. International Agency for Research on Cancer, 2014, Cancer Base No. 11. (5) Gibbs, S. L. Near Infrared Fluorescence for Image-guided Surgery. Quant Imaging Med Surg 2012, 2, 177-187. (6) Hong, G.; Antaris, A. L.; Dai, H. Near-Infrared Fluorophores for Biomedical Imaging. Nat. Biomed. Eng. 2017, 1, 0010. (7) Antaris, A. L.; Chen, H.; Cheng, K.; Sun, Y.; Hong, G.; Qu, C.; Diao, S.; Deng, Z.; Hu, X.; Zhang, B.; Zhang, X.; Yaghi, O. K.; Alamparambil, Z. R.; Hong, X.; Cheng, Z.; Dai, H. A Small-Molecule Dye for NIR-II Imaging. Nat. Mater. 2015, 15, 235-242. (8) Liu, X.; Braun, G. B.; Qin, M.; Ruoslahti, E.; Sugahara, K. N. In Vivo Cation Exchange in Quantum Dots for Tumor-Specific Imaging. Nat. Comm. 2017, 8, 343-356. (9) Chen, G.; Qiu, H.; Prasad, P. N.; Chen, X. Upconversion Nanoparticles: Design, Nanochemistry and Applications in Theranostics. Chem. Rev. 2012, 114, 5161-5214. (10) Chen, D.; Mellman, I. Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity 2013, 39, 1-10. (11) Banys, M.; Müller, V.; Melcher, C.; Aktas, B.; Kasimirbauer, S.; Hagenbeck, C.; Hartkopf, A.; Fehm, T. Circulating Tumor Cells in Breast Cancer. Clin. Chim. Acta. 2013, 423, 39-45. (12) Murlidhar, V.; Reddy, R. M.; Fouladdel, S.; Zhao, L.; Ishikawa, M. K.; Grabauskiene, S.; Zhang, Z.; Lin, J.; Chang, A. C.; Carrott, P. W. Poor Prognosis Indicated by Venous Circulating Tumor Cell Clusters in Early Stage Lung Cancers. Can. Res. 2017, 77, 5194. (13) Liu, J. N.; Bu, W.; Shi, J. Chemical Design and Synthesis of Functionalized Probes for Imaging and Treating Tumor Hypoxia. Chem. Rev. 2017, 117, 6160-6224. (14) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530-10574. (15) Kim, J.; Tung, C. H.; Choi, Y. Smart Dual-Functional Warhead for Folate ReceptorSpecific Activatable Imaging and Photodynamic Therapy. Chem. Commun. 2014, 50, 10600-10603.

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