Metastatic and non-metastatic melanoma imaging using Sgc8-c aptamer PTK7-recognizer

Melanoma is one of the most aggressive and deadly skin cancers, and although histopathological criteria are used for its prognosis, biomarkers are necessary to identify the different evolution stages. The applications of molecular imaging include the in vivo diagnosis of cancer with probes that recognize the tumor-biomarkers specific expression allowing external image acquisitions and evaluation of the biological process in quali-quantitative ways. Aptamers are oligonucleotides that recognize targets with high affinity and specificity presenting advantages that make them interesting molecular imaging probes. Sgc8-c (DNA-aptamer) selectively recognizes PTK7-receptor overexpressed in various types of tumors. Herein, Sgc8-c was evaluated, for the first time, in a metastatic melanoma model as molecular imaging probe for in vivo diagnostic, as well as in a non-metastatic melanoma model. Firstly, two probes, radio- and fluorescent-probe, were in vitro evaluated verifying the high specific PTK7 recognition and its internalization in tumor cells by the endosomal route. Secondly, in vivo proof of concept was performed in animal tumor models. In addition, they have rapid clearance from blood exhibiting excellent target (tumor)/non-target organ ratios. Furthermore, optimal biodistribution was observed 24 h after probes injections accumulating almost exclusively in the tumor tissue. Sgc8-c is a potential tool for their specific use in the early detection of melanoma.

www.nature.com/scientificreports/ production and no batch to batch variability 32,33 . They have physicochemical properties, such as stability at different temperatures and pH 34,35 , and can be easily modified to continue improving their biological stability and pharmacokinetics 26,36,37 .Furthermore, aptamers molecular weight (~ 15000 Da) and charge provide rapid penetration into target tissues and elimination from the body 31,38 . Also, aptamers do not generate immunogenicity or toxic effects. Therefore, the characteristics of aptamers provide great advantages for their use in the development of new molecular imaging agents 39 . Previously, we have modified the Sgc8-c aptamer to generate molecular imaging probes in the diagnosis of cancer [39][40][41][42] . The Sgc8-c aptamer is DNA (41 nt) and selectively recognizes the PKT7 receptor with a K d = 0.78 nM 10,43 . We have developed potential molecular imaging probes in different tumor models, through in vitro and in vivo evaluation, with an emphasis on hematological diseases. In this sense, radiolabelled probes (Sgc8-c-NOTA-67 Ga, Sgc8-c-DOTA-67 Ga, Sgc8-c-HYNIC-99m Tc) have been reported to have better tissue penetration and ability to accurately measure tissue, and as a result, it allows quantitative images of the whole body [39][40][41][42] . Likewise, the fluorescent probe (Sgc8-c-Alexa647) allows the generation of optical images in the near infrared region with little interference, achieving optimal contrasts due to the molecules present in the tissues, that do not exhibit high absorption in that spectral region 39,40 . Different versatile platforms have been reported, where the conjugation of Sgc8-c aptamer enhances the cellular uptake capability of the material, achieving specific images in vivo [44][45][46] , even in deep tissues 45 . These features are very useful to perform, for example, guided surgeries in real time 47,48 . Using the appropriate imaging probes, it is even possible to identify metastases 49 . Recently, we have studied these probes as imaging tools in a melanoma model, obtaining interesting results regarding tumor uptake 39 . Moreover, the sensitive and effective detection of PTK7 may represent a good strategy in the early diagnosis of melanoma. Based on this, the present work evaluates for the first time two potential different probes developed with of Sgc8-c in the metastatic melanoma model. Likewise, it also delves into another non-metastatic melanoma model, in order to optimize biological control methods, both biodistribution and imaging, achieving a sensitive and effective detection of PTK7 with these probes.

Results and discussion
PTK7 expression in B16F10 cells. Firstly, we evaluated the presence of PTK7 in metastatic melanoma B16F10 cells by flow cytometry and Western blot, as it was previously evaluated in non-metastatic B16F1 melanoma cells 39,40 . Flow cytometry assays performed with a commercial anti-PTK7 antibody as probe revealed that approximately 40% of the B16F10 cells expressed detectable levels of PTK7. Of note, near to 80% of the positive control CCRF-CEM cells stained for PTK7, while the negative control, U87MG cells, showed null signal (Fig. 1A). Additionally, the Western blot studies confirmed the presence of PTK7 receptor on B16F10 cells (Fig. 1B, full-length gels are shown in Fig. S1 in Supporting Information). In this assay, it was observed that the probe also recognizes two fractions of PTK7. It has been reported in different cell lines that this receptor has different cleavage sites, which generate different matching cell-associated forms 13,50 . In vitro binding studies. As it was previously described 39,40 , Sgc8-c-NOTA-67 Ga and Sgc8-c-Alexa647 probes were stable, according to RP-HPLC and gel electrophoresis results, in water up to 75 °C for 30 min of incubation and in addition the integrity of these conjugates under different storage conditions was confirmed. The observed electrophoresis profiles were consistent with the absence of low molecular weight fragmentation. Furthermore, for the precursor Sgc8-c-NOTA, the probe Sgc8-c-Alexa647 and as well as the original aptamer Sgc8-c-NH 2 , functional stabilities in DNase I, after 15 min and 2 h of incubation was studied observing similar level of degradation. After 15 min of incubation with the enzyme, it was found that Sgc8-c-NOTA was more resistant to degradation than the fluorescent probe (Sgc8-c-Alexa647). After 2 h, for all the samples, there was still aptamer without degradation. The behavior on fetal bovine serum (FBS) was also studied resulted in > 90% serum protein binding, and 66% instability 40 .
Two different strategies were employed to analyze the ability of Sgc8-c probes to interact to B16F10: the use of the radiolabelled probe (Sgc8-c-NOTA-67 Ga) that can be measured byGamma counter system, and the use of the fluorescent-probe (Sgc8-c-Alexa647), which allows analysis by flow cytometry and Western blot.
The results showed that the Sgc8-c-NOTA-67 Ga probe binds to the B16F10 cell line. It was found that the binding percentage increased with time with significant differences at 4 h of incubation (Fig. 2). Regarding the blocking test of cells with the unlabeled aptamer, it was observed that the percentage of binding to the labeled probe decreases (compare 2 h of incubation and blocking incubation, p < 0.05, Fig. 2) indicating that there was a competition between both compounds confirming the probe interaction with PTK7.
For fluorescent flow cytometry assays, melanoma B16F10 cells were incubated with different concentrations of Sgc8-c-Alexa647. Figure 3 shows the percentage of positive cells and the specific mean fluorescence index (MFI). Results showed that the percentage of PTK7 positive cells is dependent on probe concentration, reaching a maximum of 80% of B16F10 cells with approximately 0.5 μM. Furthermore, the MFI indicates the amount of the probe that binds to PTK7, more precisely, the abundance of proteins at individual population cell level 51 , thus this suggests that the B16F10 cell line expresses significant high levels of the PTK7 receptor (Fig. 3).
According to cytometry analysis, saturation concentration was reached at 0.3 μM of aptamer without achieving 100% of the B16F10 cells. This phenomenon could be explained by different cell division and differentiation stages in cell culture. In addition, as mentioned above, a molecular cleavage phenomenon has been described upon Sgc8-PTK7 interaction 46 . If the cleaved PTK7 receptor had been excreted into the supernatant of the medium, as result of cleavage mechanism of the aptamer-PTK7 complex, it would be the cause of not reaching saturation when the process was analyzed by flow cytometry. Then, if the cleaved PTK7 receptor is excreted into the supernatant of the medium, its detection by flow cytometry would not be possible. However, all experiments were performed in ice so the chances of cleavage are almost null. Besides, Western blot studies using supernatants www.nature.com/scientificreports/ rejected this hypothesis. The results indicated that both the antibody and the fluorescent probe only recognize proteins from the cell pellet ( Fig. 1B) showing that there was no detectable molecular cleavage in the supernatant. Based on these results, we propose the hypothesis that the rapid complex internalization does not allowing saturation due to the absence of the membrane receptor 53 . For this reason, fluorescent confocal microscopy studies were performed to demonstrate the internalization of the receptor (see below).
In vivo binding studies. In order to perform the in vivo biological studies we first analyzed the ability of the Sgc-8-c-Alexa647 probe to recognize in vivo the PTK7 presence in target organs. For this, flow cytometry studies were performed on B16F10-tumors, and liver, spleen and bone marrows as negative controls. The results showed that the fluorescent-probe marked, from early times, tumor cells al high levels, while in the rest of the non-target organs the percentage of positive cells was very low (Table 1), showing that the probe binds specifically to the PTK7 receptors that are on melanoma cells. In these studies, it is interesting to observe that the union of the probe to the tumor cells is maintained for a longer time than in the other non-target organs, without observing a significant tendency. This could be due to tissue homogenization to analyze the cells by flow cytometry, because everything that is retained in the extracellular matrix or contained in the different tissues is Confocal microscopy. Another mechanism proposed for Sgc8-PTK7 interaction is complex internalization 47 . For this reason, fluorescent confocal microscopy studies were performed to demonstrate the   www.nature.com/scientificreports/ internalization of the receptor on the studied tumor cells. To perform confocal microscopy assays, B16F10 tumor cells were incubated with the Sgc8-c-Alexa647 probe during different times. At early time point, it was evident that the Sgc8-c-Alexa647 probe was internalized by the cells (Fig. 4).
Since it was observed that Sgc8-c-Alexa647 is internalized, we analyzed if the probe was also co-localized within the endosomes like other aptamers-probes 53 . For that, tumor cells were incubated with the fluorescent probe for different times and subsequently the early endosomes were analyzed. Sgc8-c-Alexa647 was observed to co-localize within endosomes in both melanoma cell lines ( Fig. 5 and Fig. S2 in Supporting Information). Similarly, it was observed that the signal from the probe showed a tendency towards polarization. This is in agreement with what has been previously seen, that PTK7 is not uniformly distributed in the cell membrane, presenting a dynamic role in cell polarization 49 . In vivo biological studies. In vivo studies using both Sgc8-c-Alexa647 and Sgc8-c-NOTA-67 Ga probes were performed in murine metastatic melanoma model. To do this, once the induced tumors were palpable, probes were i.v. administered, and imaging and biodistribution studies were performed at different times after injection. The results showed interesting characteristics regarding the uptake of the probes in the tumor (Figs. 6, 7). Rapid tissue penetration was visualized, with tumor retention of both probes (Figs. 6, 7). Using the fluorescent probe, a tumor uptake of 32.9 ± 3.5% was observed at 2 h post-injection, increasing significantly at 24 h (42.4 ± 1.5%) and at 48 h (50.3 ± 1.6%) (Figs. 6A, 8). At 2 h post injection of the fluorescent probe, 33.2 ± 15.6% was eliminated in urine, decreasing to 42.6 ± 13.8% at 24 h post-injection (Fig. 6). No significant signal was observed in non-target organs (Fig. 6A). A similar tendence was observed in the non-metastatic melanoma model (Fig. S3).
The radiolabeled probe showed an increasing tumor uptake 24 h after injection. At 2 h post-injection, a tumor uptake of 8.4 ± 1.6%ID/g was observed, increasing significantly at 24 h (28.8 ± 6.2%ID/g). Then, at 72 h after injection, tumor uptake decreased slightly (17.7 ± 0.1% ID/g) (Fig. 6B). Urinary excretion was observed at early time points. A value of 9.1 ± 4.9% ID was observed at 0.5 h post-injection, which decreased to 4.2 ± 1.6%ID at 2 h (Fig. 6). These data were determined by the renal values that were due to the elimination of the probes or their metabolites (Figs. 6B, 8). After 24 h post-injection, it was possible to distinguish signals from liver and intestines in the in vivo and ex vivo images, considering hepatobiliary metabolism for later time points (Figs. 6B,  9). However, non-significant signal comes from other organs, which allowed us to observe a clear signal from the tumor (Fig. 9).
Differences in signal values between organs and tumor were evident with both probes, resulting in an optimal tumor/non-tumor organ ratio, having a significant increase over time (Fig. 7). Mainly at 48 h post-injection with radiolabeled probe in the metastatic model, generated by B16F10 cells, showed a tumor/muscle ratio of 35.5 ± 13.7 ( Fig. 7B and Fig. S4 for B16F1 model in Supporting Information), which is an excellent value to consider in vivo images. Likewise, it was observed that tumor uptake increased significantly with time in both models (Fig. S5 in Supporting Information). However, it was observed that the tumor uptake was slightly higher in the metastatic melanoma model, generated with the B16F10 cell line, than with the B16F1 non-metastatic melanoma model ( Fig. 6 and Fig. S5 in Supporting Information). This difference could be explained by an increase in the expression of PTK7, since it has been seen that in metastatic melanomas, this receptor is one of the tyrosine kinases that participates in the positive regulation of the formation and function of the invadopodia 9 . Also, this difference in www.nature.com/scientificreports/ the distribution of the probes, which is also observed in the images obtained by confocal microscopy, could be affected by a structural heterogeneity in the tumor vasculature, since it has been seen that the models generated with the B16F10 line show a significant improvement in vascular density 50 .
In vivo blocking studies with Sgc8-c-NH 2 showed a statistically significant decrease of tumor signal for the fluorescent probe ( Fig. 6 and Fig. S3, Supporting information), confirming specific probe training. These assays also reproduce the results seen in hematological models 38 , indicating that there was competition between the probe and the unlabeled aptamer. In addition with the results of the in vivo binding study (Table 1), we confirm the in vivo specific interaction of the probe with PTK7.

Conclusions
The results described herein together with our previous studies 39,42 , showed that both probes developed with the Sgc8-c aptamer are potential tools for their specific use in the early detection of melanoma. The results of in vitro studies were consistent with those obtained for biodistributions and the in vivo imaging. We obtained interesting characteristics related to the uptake of the probes in the tumor, with optimal tumor/non-target organs ratios, which are global benchmarks that encourage us to continue working on this. Although we found the best ratios at 24 h, other acquisition time points can be evaluated to find a better one. Like antibodies, aptamer-based probes are feasible in the clinic. One of most remarkable results is the low backgrounds versus high uptake in tumor which is very good and convenient. These methodologies applied here allowed us to detect differences in the expression of the tumor marker PTK7 in two different melanoma tumor models. However, it should be studied in depth whether this difference in the expression of PTK7 is really involved in signaling pathways that, consequently, grants greater metastatic power to cells.
The optimal tumor uptakes of the probes in metastatic melanoma, make them promising tools to facilitate in vivo diagnosis and thus to select an appropriate therapy. These probes could also be advantageous for www.nature.com/scientificreports/ developing intraoperative imaging devices, combined or not, the properties of both probes for use in guided surgeries; identifying and pointing out the tumor margins, helping in the surgical resection of tumors and even helpful in detecting metastases 42,51 . A deepened study with the metastatic melanoma model is currently in progress with the aim to improve the use of probes in early diagnosis that allows the selection of an efficient and personalized therapy, and even for monitoring after remission.

Confocal microscopy.
To perform confocal microscopy assays, 1 × 10 5 cells from the B16F10 and B16F1 tumor cell lines were grown on round glass coverslips (12 mm) inside 24-well plates. These cells were incubated with the Sgc8-c-Alexa647 probe (10 μg, 0.8 nmol) for different times (2, 4, and 16 h). Cells were washed with sterile PBS pH 7.4 and fixed with 4% paraformaldehyde. Subsequently, the coverslips were placed in a humid chamber, the cells were blocked with 2% bovine serum albumin in PBS for 20 min at RT and then they were blocked for an additional 15 min, also at RT, with the same solution but adding Triton (0.3%). Cells were incubated for 1 h at RT with the Hoechst 33342 nuclear marker (1:100, Immuno Chemistry Technologies, LLC) and with the WGA-green membrane marker (1:100, thermofisher scientific, USA). They were washed three times with PBS pH 7.4 and three more times with mili Q water. The coverslips were mounted with ProLong ® (thermofisher Scientific, USA) and the images were acquired in the confocal microscope LEICATCS-SP5-DMI6000 (HeNe laser, 10 mW: 633 nm). To determine if the probe was internalized endosomically, we followed the same protocol as before, incubating for 0.5, 2 and 4 h and instead of using a membrane marker, the early endosomal marker Rab5 (rabbit, 1:100, C8B1 mAb 3547, Cell Signaling Technology, USA) was used. The secondary antibody anti-rabbit IgG, Alexa 488-conjugated (goat, 1:500, ab 150077 Abcam, USA) was used.
In vivo biological studies. Animals www.nature.com/scientificreports/ pellet diet and given water ad libitum and were used after a minimum of 3 days acclimation to the housing conditions. Animals were monitored daily, recording their behavior and the presence or absence of tumor. Tumor location and volume was recorded, checking that they did not exceed a diameter of 5 mm. Isoflurane was used for anesthesia and at the end of the experiments the animals were sacrificed by cervical dislocation.
Binding studies for fluorescent-probe. For this assay 2.5 × 10 5 cells/100 mL of the B16F10 cell line were injected subcutaneously into the right flank of C57BL/6 mice. Once tumors were palpable (10-12 days), the Sgc8-c-Alexa647 probe (25 μg, 2 nmol) was injected intravenously (i.v.) through the tail vein. At 0.5, 2 and 24 h post injection, mice were sacrificed to obtain the tumor, liver, spleen and marrow derived from the femur. Organs and tissues were disaggregated by passing through a cell strainer 70 μm (BD Bioscience) and resuspended in sterile PBS pH 7.4. For each sample, 10,000 events were detected using the same laser, detector, and equipment mentioned above. The test was done in quintupled. Data were analyzed using FACS Diva and FlowJo software.
Imaging and biodistribution. To generate the melanoma tumor model murine cells B16F1were inoculated in the C57BL/6 mice, as described above for B16F10. Once melanomas were palpable, the Sgc8-c-Alexa647 probe (25 μg, 2 nmol) was injected i.v. and at 0.5, 2, 24 and 48 h post-injection (n = 5 per time group), mice were sacrificed. Ex vivo images of organs (liver, heart, lungs, spleen, kidneys, thyroid, muscle, bone, blood and tumor) were acquired using the imaging equipment above mentioned, with the X-ray and fluorescence model. The results were expressed in ROI and the tumor/blood and tumor/muscle ratios were calculated. In vivo specificity was evaluated in a competition test. One group of mice (n = 5) was first i.v. injected with Sgc8-c-NH 2 in excess of 5 times more than the probe. After 30 min post-injection, the same mice were i.v. injected with the Sgc8-c-Alexa647 probe. After 2 h mice were sacrificed, images acquired and organs weighed. Similar evaluation was performed for the radiolabelled probe. Sgc8-c-NOTA-67 Ga was i.v. injected (~ 1850 kBq) and biodistribution of the probe was followed until 72 h post-injection. Over time, the animals were anesthetized with isoflurane to perform the images in vivo. Live images were acquired for 0.5, 2, 24, 48 and 72 h after injection by X-rays andGamma modalities in the imaging equipment. For the biodistributions animals were sacrificed, images were acquired and the levels of radioactivity in the tissue were measured using a Gamma counter (PC-RIA MAS, Stratec). Radioactivity levels were expressed as percentage of injected dose per organ gram (%ID/g) and injected dose (%ID). Competitive blocking assays were also performed, injecting mice first with 0.5 nmol of Sgc8-c-NH 2 and after 30 min re-injected with Sgc8-c-NOTA-67 Ga and at 2 h the imaging and biodistribution studies were performed.
Statistical analysis. Statistical analysis was performed using the Student's t test because the data are independent and have a normal distribution. The p values of significance are indicated in each figure.