NH₂-terminated Fe₃O₄ NPs were synthesized using a previously reported protocol (Su et al., 2016). The as-synthesized NPs exhibit a clear morphology and uniform size distribution, confirmed by high-resolution transmission electron microscopy (HR-TEM) analysis, as shown in Figure 1A. The average size of the NPs is estimated to be around 6 nm. The crystallinity and phase formation of NPs was confirmed by XRD analysis, explicitly demonstrating the successful formation of Fe₃O₄ NPs. Figure 1B shows peaks at 30.1°, 35.5°, 43.1°, 53.5°, 57.4°, and 62.5°, which are respectively indexed to reflections from the (220), (311), (400), (422), (511), and (440) faces of the inverse spinel iron oxide (JCPDS No. 19-0629). The NH₂ functional group on the NP surface result in their hydrophilic nature and facilitates an even dispersion in an aqueous medium. The -NH₂ functional group on the NPs also facilitates further conjugation of targeting ligands on the NP’s surface. On the other hand, non-specific and insufficient delivery of GEF to the therapeutic site is a critical issue in the treatment of lung cancer. To address this issue, we first synthesized the GEF drug via a three-step process, and the obtained products were confirmed by ¹H NMR analysis (the synthesis process is shown in supporting information, steps 1-3). Before the conjugation of GEF on IONPs, we modified the GEF structure with the terminal carboxyl group by introducing/substituting the 6-propylmorpholino group on one side of the carbon chain of GEF (see step 4 in supporting information) which is the weakest binding with kinase. The other side of GEF remains the same with the presence of active binding site 1-N group on the quinazoline ring. Successful modification of GEF was confirmed by ¹H-NMR analysis (Supplementary Figure S1). Finally, -NH₂ functionalized Fe₃O₄ NPs were successfully conjugated with the terminal group of–COOH on modified the GEF (mGEF) drug via 1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) coupling. The schematic representation of the conjugation procedure is shown in Figure 1C. Successful formation of mGEF conjugated Fe₃O₄ NPs (mGEF@Fe₃O₄ NPs) was confirmed by UV-visible absorption spectra analysis. Absorption spectra of mGEF are specifically located at 340 nm wavelength whereas in bare NPs there is no significant absorption at 340 nm. In contrast, after modifying the Fe₃O₄ NPs with mGEF (mGEF@ Fe₃O₄ NPs), we observed significant absorption at 340 nm wavelength which strongly confirms the successful conjugation of mGEF on NPs (Supplementary Figure S2). Subsequently, different concentrations (low to high) of mGEF were added to Fe₃O₄ NPs and we monitored the changes in absorbance. Results reveal the significant enhancement of absorption intensity at 340 nm wavelength by increasing the mGEF concentration on NPs (Supplementary Figure S3). In addition, Iron-based NPs are proven T2-weighed MRI contrast agents and display excellent MR contrast abilities both in vitro and in vivo (Shen et al., 2017; Thangudu et al., 2022b; Thangudu et al., 2023). To verify the contrast enhancement efficiency of present synthesized-Fe₃O₄ NPs, T2-weighted MRI analysis was performed with a 7.0 T MRI scanner. The samples were serial diluted with various concentrations of Fe₃O₄ NPs and suspended in deionized water. Both the gradient-echo-based and spin-echo-based T2-weighted imaging showed significant enhancement in a concentration-dependent manner. As the concentration of NPs increased, the samples grew increasingly dark, corresponding to the enhanced T2 contrast compared to the deionized water alone. We also verified the MR activities of mGEF@Fe₃O₄ NPs, and noticed that the MR contrast abilities of mGEF-Fe₃O₄ NPs slightly less than bare Fe₃O₄ NPs alone, which may be attributed due to the conjugation of mGEF on the NPs surface possibly covering the NPs surface and altering the magnetic field-water interaction, resulting in a slight decrease in the paramagnetic effect (Liang et al., 2016).

Following the effective synthesis of mGEF@Fe₃O₄ NPs, in vitro studies were carried out to assess the therapeutic efficacy of mGEF@Fe₃O₄ NPs for treating non-small cell lung cancer. The cytotoxicity of NPs was investigated prior to the in vitro therapeutic evaluation. Results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity experiments show that cancer cells’ in vitro viability is unaffected by exposure to Fe₃O₄ NPs at concentrations ranging from 0.257 μM to 2570 μM (Figure 2A). Cytotoxicity results indicate that the as-synthesized Fe₃O₄ NPs are biocompatible. Previous work also notes that Fe₃O₄ NPs are biocompatible and have been investigated in several biomedical applications (Martins et al., 2021; Montiel Schneider et al., 2022). Notably, GEF is a tyrosine kinase inhibitor that has been given clinical approval to treat NSCLC (Kitazaki et al., 2005; Kazandjian et al., 2016). As a proof of concept, we conducted in vitro cell survival tests utilizing mGEF@Fe₃O₄ NPs to further demonstrate the targeting capability of GEF. To determine the targeting capabilities of mGEF, we used two distinct cell lines. PC9 is one of the NSCLC cell lines most vulnerable to GEF, while EGFR wild-type cell lines like A549, CL1-10, and CL1-5 are resistant to GEF. As a result, modified GEF successfully inhibited the PC9 cell survival and IC 50 was estimated to be around 5 μM, indicating the good targeting ability of mGEF (Figure 2B). In other cell lines, cell viability remains unchanged under the same circumstances due to GEF resistantance. Due to the fact that the modified drug was dissolved in DMSO for cell viability tests, we also examined the same concentration of DMSO by itself to determine whether or not it had any negative effects on the cells. The results showed that the concentration of DMSO used had no harmful effects on cells and caused no changes to cell viability (Supplementary Figure S4). The drug efficiency of mGEF@Fe₃O₄ NPs was then verified in PC9 cells via MTT assay. As shown in Figure 2C, cell viability of PC9 cells drastically decreased after treatment with mGEF@Fe₃O₄ NPs and the estimated IC 50 value is 2.0 μM. Further we have evaluted the IC50 value of mGEF@Fe3O4 NPs and mGEF drug (Supplementary Figure S5). Precisely, the inhibition efficiency of PC9 cells on mGEF@Fe₃O₄ NPs and modified GEF drug alone as follows 2 μM and 500 nM respectively, indicating that drug on NPs shown good therapeutic response similar like mGEF alone due to the rapid release of drug on NPs. We then evaluated the therapeutic effectiveness of Fe3O4@mGEF on PC9 and A549 cancer cells. As anticipated, increased mortality of PC9 cells was seen on mGEF@Fe3O4 NPs under the same experimental circumstances. As a result of A549s resistance to the mGEF@Fe3O4 NPs even at higher concentrations (100 μM), there was no significant cell inhibition (Figure 2D). GEF primarily blocks the tyrosine kinase domain and suppresses signal transmission. These tyrosine kinases will, however, be found inside of cells. Therefore for effective cell inhibition, the nano platform must infiltrate the intracellular setting and deliver the drugs. Consequently, we used Perls’ blue staining assay to determine whether or not the mGEF@Fe₃O₄ NPs were successfully endocytosed by the cells. As shown in Figure 2E; Supplementary Figure S6, we observed the presence of Fe₃O₄ NPs inside the PC9 cells after treatment with mGEF@Fe₃O₄ NPs. mGEF@Fe₃O₄ NPs began to transport into the intracellular domain after 4-h incubation and started to accumulate more NPs inside the cells over the prolonged incubation time. The results demonstrate that the current mGEF@Fe₃O₄ NPs were successfully endocytosed into the cell and were primarily distributed around the cell membrane, making it possible to block the tyrosine kinase. Mechanistic experiments were then carried out to further understand how exposure to mGEF@Fe₃O₄ NPs caused PC9 cells to perish. It is widely known that the primary cause of GEF cell death in EGFR L858R mutant non-small lung cancer is apoptosis (Tracy et al., 2004). In early apoptotic cells, membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment (Mariño and Kroemer, 2013). Annexin V is a Ca²⁺-dependent phospholipid-binding protein that has a high affinity to bind with exposed PS (Vermes et al., 1995). In late apoptosis, the cell membrane was damaged and became permeable, allowing the nucleic acid dye (Propidium iodide, PI) to be intercalated in nucleic acid. Thus, to distinguish the mechanism of cell death induced by mGEF@Fe₃O₄ NPs, we performed Annexin V-FITC and PI fluorescence staining analysis on gefitinib-sensitive PC9 cells as well as gefitinib resistant A549 cells. As shown in Supplementary Figure S7, the cell density of PC9 showed no significant difference from the control group up to treatment with concentrations of 300 nM of mGEF@Fe₃O₄ NPs. In contrast, increasing the concentration of mGEF@Fe₃O₄ NPs to 100 μM resulted in increased PC9 cell death. On the contrary, we observed no significant response to mGEF@Fe₃O₄ NPs for A549 cells, even with dosages of up to 100 μM (Supplementary Figure S8). Although the morphology of the PC9 cell looks undamaged after treatment with 300 nM concentration of NPs, it processed in early apoptosis, as shown in the Annexin V-FITC image. For both Annexin V-FITC or PI staining, A549 cells were still active with reduced fluorescence which respectively represented apoptosis and necrosis. Results of fluorescence microscopy studies verified the mGEF@Fe₃O₄ NPs lead cell apoptosis and that it is specific to PC9 cells.

Thereafter, mGEF@Fe₃O₄ NPs’ MR imaging capabilities were assessed both in vitro and in vivo. First, to assess the MR contrast effect of mGEF@Fe₃O₄ NPs within the cells, various concentrations of mGEF@Fe₃O₄ NPs were incubated with PC9 cells for a given time for the MR imaging experiments. As shown in Figure 3A, no significant changes were noticed in the case of cells incubated with mGEF and cells alone. In contrast, cells treated with mGEF@Fe₃O₄ NPs darken significantly than other control groups due to the T2 effect. Moreover, increasing concentrations of mGEF@Fe₃O₄ NPs dramatically changes the signal intensity of the T2-weight phantom images and increases the darkness. The difference in signal intensity reached 35% (at low conc.) and 87% (at high conc.) respective to the control group, as shown in Figure 3B. In vitro contrast ability studies demonstrated the successful internalization of mGEF@Fe₃O₄ NPs into cancer cells, indicating feasibility for T2-enhanced MR imaging applications. Furthermore, in vivo MR imaging was performed by establishing the xenograft PC9 tumor-bearing animal model by subcutaneously injecting 4 ×10⁶ PC9 cells (in 0.1 mL PBS) at the mouse’s left back (Supplementary Figure S9). Two weeks after tumor implantation, mGEF@Fe₃O₄ NPs (30 mg OF [Fe]/kg) were injected intravenously via the tail vein. Thereafter, in vivo MR imaging was performed at different time points (pre- and post 9 h-injection of mGEF@Fe₃O₄ NPs). Figure 3C shows the MR images of mice (n = 3) pre- and post-injection of mGEF@Fe₃O₄ NPs. 9 h post-injection, the signal intensity in the tumor was significantly lower than in the control group, which indicates that the accumulation of NPs at the tumor site corresponding to the successful delivery of chemo drug to the tumor increased locally. As a result, contrast intensity gradually decreased correspondingly to the T2 effect by increasing the post-injection time of mGEF@Fe₃O₄ NPs. The signal intensity at the tumor region was decreased to 15% after 4–9 h of post admisnstartion of NPs, and after 24 h it gradually dropped to 23% (Supplementary Figure S10). Even though in vivo imaging showed a significant accumulation of Fe₃O₄@gefitinib NPs at the tumor, the degree of enhancement is not as significant as expected, probably due to the significant differences of contrast between the inner and outer regions of the tumor 9 h post-injection of mGEF@Fe₃O₄ NPs. The negative enhancement of the inner region was not as obvious as the outer region, and the inner was brighter than the outer tumor in T₂-weighted MR images. Thus we segmented the tumor into outer and inner regions and measured the contrast change at the different time points (Figures 4A, B). After 9 h of mGEF@Fe₃O₄ NPs injection, the signal intensity of the outer tumor region decreased by 12% whereas the inner tumor region only decreased by 4%. The inner region was proton-rich so the signal intensity was higher than around the tissue in the T₂-weighted imaging. According to our hypothesis, the inner region had undergone necrosis. Cells swelled and even disintegrated after the intracellular contents were released, so the images showed hyperintensity in T2-weighted imaging. Consequently, it was difficult to deliver the mGEF@Fe₃O₄ NPs into the inner region where vasculature was lacking. As proof of concept, we analyzed and compared the histology of the outer and inner tumor regions. As shown in Supplementary Figures S11, S12, cells of the inner tumor (left) were shrunken with minimal amounts of iron, whereas the cells located in the outer region (right) retained their shape with intensive iron deposits. H&E staining analysis of the tumor region (Supplementary Figure S13) revealed distinct differences between the morphologies of the inner and outer regions, where the cells of the outer region were crowded and intact, while those of the inner region were arranged loosely and shrunken, indicating that the inner region cells underwent greater necrosis.

To further investigate he differences between outer and inner tumor regions, we also performed PET/CT scan imaging to obtain functional information such as staging, glucose metabolism and cell cycle physiology. We separately used the [¹⁸F] fluorodeoxyglucose (FDG) and [¹⁸F] fluoro-L-thymidine (FLT) as a tracer for PET scanning (Rasey et al., 2002; Ullrich et al., 2008). The tracer fluorine-18 [¹⁸F]) fluorodeoxyglucose (FDG) is a glucose analog that is taken up by glucose-using cells and phosphorylated by hexokinase, the mitochondrial form of which is greatly elevated in rapidly growing malignant tumors (Czernin et al., 2013; Kawada et al., 2016). The [¹⁸F]-fluoro-L-thymidine ([¹⁸F]FLT) is a specific marker to measure cellular proliferation in vivo. As an analog substrate of thymidine, [¹⁸F] FLT is phosphorylated by thymidine kinase 1 which is a cytosolic enzyme synthesized when proliferating cells enter the S-phase for DNA synthesis. First, we measured the maximum standard uptake value (SUV) of the tumor and the normal tissue using dynamic PET imaging (Supplementary Figure S14). The time-activity curve (TAC) shows the SUV of normal tissue decreases over time, while the tumor uptake remained consistent 0–120 min post-injection, and the contrast between the tumor and normal tissue is gradually enhanced. The obvious difference is found at 120 min after injection, indicating the suitable image acquisition time for distinguishing tumor from normal tissue is 60–120 min after tracer injection. Figures 4C, D show the[¹⁸F] FDG and [¹⁸F] FLT PET/CT scan results on PC9 NSCLC mice model. In [¹⁸F] FDG or [¹⁸F] FLT images, the tumor site presented a circular donut-shaped hot spot (tumor region indicated with an arrow). The γ-ray emission means in vivo biodistribution of the tracer, and the radioactivity of the inner region was so low even near the background, indicating that the tracer could not be transported into the inner side or the dead cell is disabled to take up the tracer. In conclusion, the inner tumor was necrosed and deficient in activity. Because of the tumor necrosis at the inner region, there was no blood vessel to deliver material including radioactive tracers or Fe₃O₄@gefitinib, sufficiently explaining that MR imaging could be enhanced only at the outer region of the tumor.

Biocompatibility plays a vital role before prior to the implementation of biomedical health applications (Thangudu et al., 2022a). In the former section, we demonstrate in vitro that the self-synthesized Fe₃O₄ NPs are nontoxic and efficiently enhanced MRI tissue contrast. Although the biocompatibility of iron oxide nanoparticles is well-known, we are still concerned with potential excretion. Following intravenous administration of Fe₃O₄ NPs, we used the 7T MRI to observe the contrast in mice liver (n = 3) at different time points up to a week (Supplementary Figure S15). The Fe₃O₄ NPs gradually accumulated in the liver, hence the average signal intensity decreased over time, the liver contrast in MRI was retrieved to pre-administration, and we can speculate that NPs accumulated in the liver were further excreted over the course of the week. Furthermore, we evaluated the biodistribution of Fe, performing histological Perl’s blue staining assay after Fe₃O₄ NPs administration via iv injection. At different post-injection time points (1.5 h, 3, 6, and 9 days) the animals were sacrificed and livers were collected, embedded in paraffin, and sectioned for Perls’ blue staining (Supplementary Figure S16). This assay will be helpful to detect iron selectively, with iron appearing blue and the cytoplasm pink. Fe deposition in the liver region was substantially increased post-1h NPs injection. After prolonged post-injection time (from 3–6 days), Fe in the liver region gradually decreased and, on the 9-day post-injection, no noticeable Fe content was observed in the liver region. These results revealed the Fe₃O₄ NPs are biocompatible and excreted after treatment without creating adverse effects on the major organs.

The preparation of NH₂—Fe ₃ O ₄ NPs was carried out as previously described (Su et al., 2016). 1 M ferric chloride hexahydrate (FeCl₃·6H₂O) and 2.0 M ferrous chloride tetrahydrate (FeCl₂·4H₂O) were prepared by separatly dissolving in 2 M HCL solution. After that, 4 mL of FeCl₃·6H₂O was mixed with 1 mL of FeCl₂·4H₂O solution and then 1 mL of a organic acid solution was added under constant stirring. Subsequently, the pH of the solution was carefully adjusted to 13 by adding 5 M NaOH followed by the proper amount of adherent to achieve -NH₂—Fe ₃ O ₄ NPs.

In a typical preparation, the modified drug (40 μM) was dissolved in 1 mL DMSO solution, followed by the addition of Fe3O4 nanoparticles (40 nM) and EDC with overnight stirring at room temperature (Su et al., 2016). The resulting products were subjected to five centrifugation/re-suspension with a DMSO solution followed by water to remove the excess or unbonded drug molecules and EDC. Finally, Fe₃O₄-drug NPs were resuspended in 0.2 mL DMSO solution and stored. For further biological tasks in vitro and in vivo, the surface of Fe₃O₄@gefitinib was modified with PEG (MW: 4000) to improve the solubility of gefitinib in water.

The toxicity of iron oxide nanoparticles was assessed by MTT assay using the same protocol as that used for drug efficiency evaluation. PC9 cells were cultured in a 96-well at an initial density of 4 ×10³ cells/well. After 24 h, different concentrations of the Fe₃O₄ nanoparticles were added to the cells with final concentrations ranging from 0.257 to 2570 μM. After 24 h incubation, the culture medium was removed and 3-[4,5-dimethylthiazol-2-yl] −2,5-diphenyl tetrazolium bromide (MTT) reagent (0.5 mg/mL, Sigma) was added, followed by another 1.5-h incubation to allow the formation of formazan dye. Next, the culture medium was removed, and the DMSO (100 μL/well) was added across the plate. Cell viability was quantified using an ELISA plate reader to acquire 570 nm optical absorbance. PC9 (human non-small-cell lung adenocarcinoma) cell lines present in this study were obtained from American Type culture Collection (ATCC).

We used both the GEF drug-sensitive (PC9) and the resistant (A549, CL1-0, CL1-0 transfect VEGF isoform 189 and CL1-5, all wild type) cell lines to assess drug efficiency. The cell lines present in this study were obtained from American Type culture Collection (ATCC).

Briefly, different cancer cells were cultured in a 96-well at an initial density of 4 ×10³ cells/well. To measure drug targeting performance, different concentrations of mGEF in 100 μL of DMSO (0.1 nM–100 μM) were added to the culture wells. After 24 h incubation, the culture medium was removed and MTT reagent (0.5 mg/mL, Sigma) was added, followed by another 1.5-h incubation to allow the formation of formazan dye. Next, the culture medium was removed and the DMSO (100 μL/well) was added across the plate. The quantification determining cell viability was using an ELISA plate reader to acquire 570 nm optical absorbance.

To measure the mGEF@Fe₃O₄ NPs drug activity, PC9 and A549 cancer cells were seeded in a 96-well plate (4 ×10³ cells/well). After 24 h, different concentrations of the mGEF@Fe₃O₄ NPs were added to the cells with final concentrations ranging from 0.0 to 100 μM, and activity levels were measured using an ELISA plate reader to acquire 570 nm optical absorbance.

The cells were seeded on the 4-well chamber slides (Millipore, Millicell EZ slide) in a density of 1.55 × 105/0.1 mL/well. After the cells were attached at the bottom of the dish (about 24 h), we added the prepared nanoparticles-drug into one well of the chamber slide. After at diffrent time of incubations, perls’ blue stain was used to observe the nanoparticles endocytosed into the intracellular domain. Before staining, PBS washed the cell twice to wash out the nanoparticle-drug at the extra-cellular domain. The cells were fixed by 4% formalin for 20 min and PBS washed 3–5 times. The protocols of Perls’ blue stain are as follows (Su et al., 2016).

The sections were incubated in a solution of 4% potassium ferrocyanide solution and 4% HCl (1:1 mixed) for 10 min. After PBS washing, the cytoplasm was stained in pararosaniline solution (1 mL 1% hydrochloric acid solution diluted in 50 mL ddH2O) for 5 min, then rinsed in PBS to wash out the remaining solution. Next, we removed the cover chamber to access the microscope slide, and sections were dehydrated in 50%, 70%, 90%, and 100% EtOH and rinsed in xylene twice.

After 96 h of drug treatment, cells were washed twice with 4°C PBS. We then prepared the mixing solution (including Annexin V binding buffer (100 μL/well) and fluorochrome-conjugated Annexin V (5 μL/well), and PI solution (10 μL/well)) and added into each well. After 15 min, the fluorescence microscopy results depicted the Annexin V-FITC in green and the PI in red.

The PC9 cells were cultured in 10 cm-diameter dishes with an initial density of 5×10⁶ cells/well. After 1 day, the Fe3O4@gefitinib nanoparticles with 150 μM and 300 μM drug concentrations were added to the cells. We also treated the modified drug and medium (as control) in the other two dishes, so there is a total of four groups all treated for 24 h. The cells were washed twice with phosphate-buffered saline (PBS) and acquired the cells by 0.25% trypsinization. The cells were placed in a micro-tube and replenished with PBS. Just before MRI scanning, we centrifuged the cells down to the bottom of the tube and put four tubes in a holder immersed in mineral oil as previously described. In image acquisition, we selected the tubes that were full of cells at the bottom. The images were performed with a T2-weighted, T1-weighted sequence.

The procedures for all animals were carried out in accordance with the guidelines of the National Research Council (United States) and the Animal Welfare Regulations of the Council of Agriculture, Executive Yuan (R.O.C.). All experimental protocols involving live animals were reviewed and approved by the Chang Gung Memorial Hospital Institutional Animal Care and Use Committee (approval number: IACUC No. 2021031802). The tumor animal model was generated by subcutaneously injecting 4 ×10⁶ PC9 cells (in 0.1 mL PBS) at the mice left back. Two weeks later, the mice were subjected to MR imaging studies.

We used a 7.0 T MRI scanner to observe the in vivo image enhancement and performed experiments by following the previously reported protocol (Su et al., 2016). The Balb/C mice (male, 25–30 g) were anesthetized with 5% isoflurane, and a 30G needle connected to a syringe with a 50 cm polyethylene tube (PE8) was inserted in the tail vein. After acquiring the pre-imaging, we injected the prepared iron oxide nanoparticles dispersed in normal saline with 10 mg [Fe]/kg dosage and additionally injected 0.5 mL saline to wash the nanoparticles left in the polyethylene tube into the vein. The images were taken immediately, and 0.5 h followed up to 1.5 h. The signal intensity of the tissue was determined using standard region-of-interest measurements with homemade image quantification software. The image-acquiring pulse sequence is TurboRARE T2 pulse sequence (TR: 4000 ms, TE: 33 ms, FA: 180 deg, Matrix size: 256 × 256 × 17, FOV: 4 × 4 cm, NEX: 3, Thickness: 1 mm).

The mice were briefly anesthetized using isoflurane gas, after which 350–390 μCi of ^[18F] FLT or ^[18F] FDG tracer was injected through the tail vein. After a 60-min conscious uptake period, we performed CT scanning and a 60-min PET acquisition, with images covering the area from the diaphragm to the coccyx (2D mode, one-bed positions). Emission data were corrected for random coincidences and scatter coincidences dead time, then reconstructed by OSEM. The matrix size was 175 × 175 pixels with a pixel size of 0.3875 × 0.3875 mm.

The paraformaldehyde-fixed paraffin-embedded organs were sectioned and mounted on glass slides, followed by drying in an oven for 1 h at 65 C. Subsequently, an iron working solution was prepared by mixing equal volumes of hydrochloric acid solution and potassium ferrocyanide solution (HT20; SigmaAldrich). Later, all the organ-coated glass slides were treated with a working solution for 10 min followed by water wash and treated with a working pararosaniline solution for 2 min. All the slices were then washed with deionized water for 2 min and then rapidly dehydrated using alcohol and xylene. Finally, images were acquired using a microscope (Olympus BX51; Yuan Li Instrument Co., Taipei, Taiwan).

After the scanning, the mouse was sacrificed and the tumor was excised for further paraffin embedding, followed by H&E staining.

All data will be presented as the mean ± standard error and compared between groups using the Student’s t-test or one-way ANOVA using GraphPad Prism 5 Software (GraphPad Software, Inc., San Diego, CA). A p-value of <0.05 will be considered statistically significant. *: p < 0.05; **: p < 0.01; ***: p < 0.001.