Visual screening and efficacy evaluation of high-performance fluorescent probe DAF-FM in esophagitis cancer transformation

Abstract

BACKGROUND

Chronic esophagitis can progress to esophageal cancer via "inflammation-dysplasia-cancer" transformation, with nitric oxide (NO) serving as a critical mediator in this process. Traditional diagnostic methods (e.g., endoscopic biopsy) for esophageal cancer transformation have low sensitivity and require long detection time, while existing fluorescent probes lack specificity and stability for real-time NO monitoring. High-performance fluorescent probes like DAF-FM, with NO-targeting ability, show potential for visual screening and efficacy evaluation but need systematic validation in esophageal cancer models.

AIM

To validate the applicability of the fluorescent probe DAF-FM for visual screening of esophageal cancer transformation, explore the underlying mechanism of NO-regulated transformation, and evaluate the probe’s efficacy in monitoring therapeutic responses.

METHODS

Laser confocal imaging and flow cytometry were used to analyze DAF-FM’s NO concentration/time-dependent fluorescence response, lysosomal targeting (via Pearson coefficient), and cytotoxicity (with cholecystokinin-8 assay) in esophageal cells. Sprague-Dawley rat esophageal cancer models (normal, esophagitis, esophageal cancer, and drug/radiotherapy intervention) were established to monitor NO dynamics and tumor volume correlation. Clinical diagnostic comparison (50 suspected patients) with endoscopic biopsy/histopathology was conducted using Kolmogorov-Smirnov test and Student’s t-test (P < 0.05). Western blot and quantitative real-time polymerase chain reaction were used to explore NO’s role in the nuclear factor-kappa B (NF-κB) pathway.

RESULTS

DAF-FM exhibited concentration/time-dependent fluorescence with NO (300 μM NO: 60-minute fluorescence intensity 458 ± 15 arbitrary units, P < 0.05) and specific lysosomal targeting (Pearson’s coefficient = 0.82 ± 0.03). It had low cytotoxicity (82.3% ± 4.1% cell viability at 50 μM). In rat models, DAF-FM showed that NO was correlated with tumor volume (R² = 0.87). Clinically, its sensitivity (92.5%) outperformed endoscopic biopsy (78.3%), with shorter detection time (30 minutes vs 48 hours, P < 0.05). Mechanistically, NO regulated transformation via the NF-κB pathway (Pearson’s coefficient = 0.78 ± 0.05 between DAF-FM and NF-κB).

CONCLUSION

DAF-FM is a feasible tool for visual screening of esophageal cancer transformation, enabling real-time NO monitoring, high-sensitivity diagnosis, and therapeutic efficacy evaluation. It provides a new approach for esophageal cancer diagnosis and mechanism research.

Key Words: DAF-FM; Fluorescent probe; Esophageal cancer transformation; Nitric oxide; Visual screening; Efficacy evaluation; Lysosomal targeting

Core Tip: This study systematically evaluated the high-performance fluorescent probe DAF-FM for visual screening and efficacy assessment of esophagitis-to-cancer transformation. DAF-FM exhibited concentration-dependent and time-dependent fluorescence responses to nitric oxide (NO), targeted lysosomes specifically (Pearson coefficient = 0.82 ± 0.03), and had low cytotoxicity (82.3% ± 4.1% cell viability at 50 μM). In Sprague-Dawley rat esophagitis cancer models, DAF-FM monitored NO changes dynamically, with results positively correlated to tumor volume (R² = 0.87) post 5-fluorouracil/radiotherapy. Clinically, it outperformed endoscopic biopsy (sensitivity: 92.5% vs 78.3%) and shortened detection time (30 minutes vs 48 hours). Mechanistically, NO regulates carcinogenesis via the nuclear factor-kappa B pathway, clarifying DAF-FM’s molecular logic in reflecting transformation stages.

INTRODUCTION

Inflammation is a recognized feature of malignant tumors, and the immune microenvironment formed by chronic inflammation is closely related to the occurrence and development of tumors. When chronic inflammation is present for a long time, this leads to a large number of inflammatory cytokines and active mediators, which cause DNA damage, genomic instability and even inactivation of base mismatch repair enzymes, further leading to overexpression of oncogenes and inactivation of tumor suppressor genes, and ultimately to malignant transformation of cells[1,2]. In the tumor microenvironment, due to the characteristics of infection, high pressure and low oxygen, there are a large number of cell chemokines, growth factors and various proteolytic enzymes in the tumor microenvironment, which can form an immune inflammatory response, which is very beneficial to the proliferation, invasion, adhesion, angiogenesis and resistance to radiotherapy and chemotherapy of tumor cells[3,4]. A large number of immunosuppressive cells, regulatory T cells, tumor-related macrophages and inflammatory factors gather in the microenvironment, which can promote the immune escape of tumor cells and accelerate tumor growth and metastasis, all of which are the final outcome in the evolution of the tumor microenvironment[5]. The transformation of inflammatory cancer has been the focus of research in recent years. The inflammatory response is accompanied by the production of nitric oxide (NO), and the progression of tumors is closely related to pyruvaldehyde and glycolysis[6]. In recent years, more and more studies have shown that inflammatory bowel disease is closely related to the risk of esophageal cancer, and the inflammatory environment has an important impact on the process of "inflammatory cancer transformation" of esophageal cancer[7,8]. In this process, the inflammatory response activates a series of signal transduction pathways, affecting cell growth, differentiation and survival, ultimately leading to tumor formation[9,10]. However, the key drivers and specific mechanisms in these processes are still not fully understood.

Therefore, the in-depth study of the inflammatory-tumor transformation process of esophageal cancer and its molecular basis are of great practical significance for the discovery of new prevention and treatment strategies.

Molecular fluorescence probes have become a useful tool in biological and medical research due to their advantages of high sensitivity, easy operation and non-destructive testing[11,12]. The NO fluorescent probe 3-amino, 4-aminomethyl-2',7'-difluorofluorescein diacetate (3-Amino,4-aminomethyl-2',7'-didiacetate, DAF-FM DA) has been commercialized and widely used for intracellular NO detection[13]. It uses fluorescein as the fluorophore and the -NH2 of o-phenylenediamine as the recognition group. NO reacts with -NH2 in DAF-FM DA to produce fluorescein derivatives containing triazole which emit strong fluorescence[14]. DAF-FM DA is cellular permeable and is catalyzed by the esterase within the cell to form DAF-FM that cannot cross the cell membrane. DAF-FM itself has fluorescence. When NO is present, DAF-FM reacts with NO to produce strong fluorescence triazole fluorescein (DAF-2T) with an excitation wavelength of 491 nm and emission wavelength of 513 nm. The fluorescence intensity of the probe can be detected by fluorescence microscopy, laser confocal microscopy, flow cytometry, enzyme labeling and other instruments[15,16]. Therefore, DAF-FM DA is a relatively sensitive fluorescent probe, and the detection limit of NO at neutral pH can reach 2-5 nM[17]. The targeting NO-specific fluorescence of DAF-FM DA provides a new approach for the accurate detection of inflammation, and therefore creates a new opportunity for the early accurate detection of the malignant risk of inflammatory cancer transformation[18].

However, due to the complex and diverse pathogenesis environment, most of the probes used for the detection of inflammatory cancer transformation have poor specificity and stability, and cannot be monitored in real time; thus, the accurate and sensitive detection of esophagitis cancer transformation is limited. Based on chemical design and screening and nanoassembly strategies, we developed the NO quantitative fluorescent probe DAF-FM diacetate (DAF-FM) with high selectivity, excellent photostability and biocompatibility across the bond energy transfer ratio to accurately monitor endogenous NO during the transformation of esophagitis cancer. Through the combination of rational design and quantitative calculation, we developed a new fluorescent dye and used it to develop a series of high-performance fluorescent probes, which successfully realized the accurate detection of relevant active molecules in the transformation process of esophagitis cancer. We used the quantitative fluorescent NO probe DAF-FM to detect NO in esophageal mucosal epithelial cells and small esophageal carcinoma. On this basis, we explored the functional characteristics of the probe DAF-FM and the in vitro imaging ability of esophagitis and esophageal cancer, analyzed the expression of NO in lysozymes, statistically analyzed the above indicators detected by the probe DAF-FM, and use this probe to further reveal the physiological mechanism of the transformation process of inflammation and cancer. We summarize and elucidate the visual screening and efficacy evaluation of DAF-FM probe in the transformation of esophagitis cancer, in order that this probe becomes an important tool for exploring the transformation mechanism of esophagitis cancer, and a promising imaging tool for the diagnosis and prevention of clinically related diseases.

MATERIALS AND METHODS

Reagents and instruments

Lysine, glycine and ascorbic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. DL-homocysteine, L-cysteine, 9,9-dioctyl-2-bromofluorene, N-acetyl-L-cysteine and pyruvaldehyde were purchased from Shanghai Maclin Biochemical Technology Co., Ltd. 4,7-dibromo2,1,3-benzothiadiazole, glutathione and NO donor salts [2 (-N,N-diethylamino)-diazene-2-sodium oxide salt hydrate] were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Other reagents were purchased from Sinopharm Group Chemical Reagent Co., Ltd. The reagents used in the experiment were all analytically pure. The solution was prepared with secondary water. Nuclear magnetic resonance carbon and nuclear magnetic resonance hydrogen were tested using a Bruker Avance III HD 400 MHz superconducting nuclear magnetic resonance instrument. The high-resolution mass spectrometer used was an Orbitrap Fusion Lumos (manufactured by Thermo Fisher Scientific). A UV-1900 UV-VIS spectrometer produced by Shimadzu Company, Japan was used in the ultraviolet absorption spectrum experiment. The fluorescence spectrum experiment adopted the FS-5 fluorescence spectrometer produced by Edinburgh Instruments, United Kingdom. A LSM710 Laser scanning confocal microscope and a BD FACSAria III sorting flow cytometer (BD Biosciences) were also used.

Cytotoxicity of probes and products

We investigated the cytotoxicity of DAF-FM at different concentrations before cell imaging. By analyzing the cytotoxicity of RAW264.7 macrophages, the concentration range of DAF-FM which can be used for cell imaging was obtained. Six concentration groups (0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM), with 6 replicate wells in each group, were included to detect the cell survival rate at different concentrations. Cell level probe imaging was then performed on esophageal mucosa cells, esophageal mucosa cells treated with an inflammatory factor (lipopolysaccharide), and both NCE and TE-1 esophageal cancer cells. Three biological replicates were included for each cell, and 5 technical replicates were set for each replicate. To optimize the imaging time of the probe, in order to achieve cell imaging under the best conditions, it was necessary to investigate the change of fluorescence intensity during confocal imaging of the probe at different times (0 minute, 20 minutes, 40 minutes, 60 minutes). Each time point was detected 3 times, and the average value was used for analysis.

Cytotoxicity test

The cytotoxicity of fluorescent probes to esophageal carcinoma cells was calculated by the cholecystokinin-8 (CCK-8) method. First, esophageal cancer cells were transferred to 96-well plates (5 × 10³ cells per well) and cultured for 24 hours in an incubator containing 5% CO₂ and 95% air at 37 °C. The medium was then replaced with fresh DMEM containing fluorescent probes (final concentrations were 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM, respectively). Six replicate wells were included for each group. After 24 hours, the DMEM medium was removed, and CCK-8 solution (5 mg/mL, 20 μL) was added for 4 hours, and then DMSO (200 μL) was added to dissolve and release the purple mezan compound. Finally, the absorbance at 490 nm was measured on an enzyme-labeled instrument. Cell survival rate was calculated according to the formula: Cell survival rate = (absorbance of experimental group - absorbance of blank group)/(absorbance of control group - absorbance of blank group) × 100%. The experiment was repeated 3 times, and the average value was used for statistical analysis.

Study on colocalization of the probe

We investigated the ability of DAF-FM probe to target lysosomes. Colocalization experiments combined 5 μM probe Mito-2 or Mito-3 with 200 nM commercial dye Mito-Tracker Green or BODIPY493/593 or Lyso-Tracker Green in HeLa cells (or HepG2 or MCF-7 cells). The cells were incubated for 20 minutes, washed twice with phosphate buffer saline, and then fresh DMEM was added for laser confocal imaging. Three replicate wells were included for each cell type. Image J software was used to calculate the colocalization coefficient (Pearson correlation coefficient). Ten fields of view were randomly selected from each sample for analysis, and the average value was obtained. A colocalization coefficient > 0.7 was considered to indicate good targeting.

Confocal imaging and flow cytometry of exogenous NO

In order to explore the imaging ability of the probe on the ratio of NO in lysozymes, following incubation of the DAF-FM probe with the cells, different concentrations of NO donor DEANO (0 μM, 50 μM, 150 μM, 300 μM) were added, the cells were incubated for 1 hour, and cell imaging was performed. Three replicate wells were included for each concentration group, and Image J software was used to quantitatively analyze the fluorescence intensity. Flow cytometry proved that the probe could quantitatively detect exogenous NO. The experiment included the above-mentioned 4 DEANO concentration groups, 1 × 10⁴ cells were detected in each group, and the experiment was repeated 3 times to draw a fluorescence intensity-concentration standard curve (R² = 0.98). For the detection of cell NO, it was necessary to select a control cell group (blank control group: No probe added; negative control group: Probe added but no DEANO added; experimental group: Probe and different concentrations of DEANO added), deduct the endogenous background NO, and analyze according to the fluorescent-concentration curve.

Efficacy evaluation experiment

Sprague-Dawley rats were used to establish esophagitis cancer models, and the animals were randomly divided into 4 groups with 10 rats per group: (1) Normal control group (n = 10); (2) Esophagitis model group (n = 10); (3) Esophagitis cancer model group (n = 10); and (4) The drug [5-fluorouracil (5-Fu), 10 mg/kg] + radiotherapy (2 Gy dose) intervention group (n = 10). The esophagitis model was established using 0.15% deoxycholic acid by gavage (10 mL/kg, once a day for 4 weeks); the esophagitis cancer model was established by continuing to administer N-methyl-N-nitrosourea (20 mg/kg) by gavage once a week for 8 weeks on the basis of the esophagitis model. The modeling success standard was achieved when a pathological section showed that the esophageal mucosa had severe dysplasia or carcinoma in situ. The DAF-FM probe was used to monitor the NO concentration changes and fluorescence intensity in the esophageal tissue of rats in each group, once a week for 4 consecutive weeks. In addition, the volume of esophageal tumors in rats was measured (volume = length × width²/2), and tumor growth was recorded. At each time point, 3 rats per group were randomly selected for tissue sampling to avoid repeated sampling bias. In addition, rat esophageal tissue samples from each group were collected, and immunohistochemistry was conducted to detect the expression of Ki-67 (proliferation index) and caspase-3 (apoptosis index), and the correlation between the probe monitoring results and tumor proliferation and apoptosis was analyzed.

Comparison of diagnostic methods

Fifty patients with suspected esophagitis cancer (28 males, 22 females; age range: 45-68 years, mean age: 56.2 ± 7.8 years) were prospectively enrolled from the Department of Gastroenterology, Quanzhou First Hospital Affiliated to Fujian Medical University between January 2025 and March 2025. All patients met the following inclusion criteria: (1) Presence of clinical symptoms (e.g., dysphagia, retrosternal pain); (2) No prior esophageal surgery or radiotherapy/chemotherapy; and (3) Willingness to undergo DAF-FM probe detection, endoscopic biopsy, and histopathological examination. Exclusion criteria included: (1) Severe liver/kidney dysfunction; (2) Autoimmune diseases; and (3) Pregnancy or lactation. DAF-FM probe detection, endoscopic biopsy and histopathological examination were performed, respectively. Using the histopathological examination results as the gold standard, the sensitivity [sensitivity = number of true positive cases/(number of true positive cases + number of false negative cases) × 100%], specificity [specificity = number of true negative cases/(number of true negative cases + number of false positive cases) × 100%], accuracy [accuracy = (number of true positive cases + number of true negative cases)/total number of cases × 100%] and detection time of the three methods were calculated for statistical analysis.

Mechanism exploration experiment

To clarify the specific mechanism of NO in esophagitis carcinogenesis and the molecular logic of DAF-FM reflecting carcinogenesis by monitoring NO, the following experiments were conducted.

Western blot detection: Esophageal cancer cells were treated with different concentrations of NO donors (0 nM, 50 nM, 100 nM, 200 nM, 300 nM) for 24 hours. Three replicate wells were included for each concentration group, and the experiment was repeated 3 times independently to ensure result reproducibility. Total protein was extracted from cells, and the expression levels of nuclear factor-kappa B (NF-κB) (p65), c-Myc, Bcl-2, and p53 proteins were detected by western blot. β-actin was used as the internal reference. The gray value of protein bands was analyzed by Image J software, and the relative expression level of each protein was calculated (relative expression level = gray value of target protein band/gray value of β-actin band).

Quantitative real-time polymerase chain reaction detection: The same NO concentration-treated esophageal cancer cells as above were used, with 3 replicate wells per group. Total RNA was extracted using TRIzol reagent (Invitrogen, United States), and RNA purity was verified by measuring A260/A280 ratio (1.8-2.0) using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). Total RNA was extracted, reverse-transcribed into cDNA, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the mRNA expression levels of NF-κB (p65), c-Myc, Bcl-2, and p53 genes. GAPDH was used as the internal reference. The relative expression level of mRNA was calculated by the 2^-ΔΔCt method.

Immunofluorescence colocalization experiment: Esophageal cancer cells were incubated with 5 μM DAF-FM probe and 1 μg/mL NF-κB (p65) primary antibody (Abcam, ab16502) at 37 °C for 2 hours, then incubated with Cy3-labeled secondary antibody (Abcam, ab6939) for 1 hour. Three replicate wells were included, and laser confocal microscopy (LSM710, Zeiss, Germany) was used for imaging. Z-stack images were acquired at 0.5 μm intervals to ensure full colocalization analysis, and ImageJ software (with Coloc 2 plugin) was used to calculate the Pearson correlation coefficient between DAF-FM fluorescence signals and NF-κB (p65) immunofluorescence signals, to analyze the association between NO (monitored by DAF-FM) and the NF-κB signaling pathway.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences 25.0 (IBM, United States). The results are expressed as mean ± SD. The difference between the two groups was determined by the unpaired student t test. In addition, one-way analysis of variance (ANOVA) with the minimal significant difference post facto test was used to compare the mean values between multiple groups, two-tailed two-factor ANOVA was used in multiple comparisons, and then Bonferroni post facto analysis was used to determine interactions. For the comparison of diagnostic methods, the χ² test was used to compare the sensitivity, specificity, and accuracy between the groups. P values less than 0.05 were considered statistically significant.

RESULTS

Imaging of the rat model of esophagitis showed that the esophageal mucosa was rough, infiltration of inflammatory cells was serious, inflammation of the lower esophageal mucosa had changed, that is, inflammatory cells such as lymphocytes and neutrophils had infiltrated, epithelial defects, submucosal tissue edema and capillary dilatation were present, and mucosal damage and ulcers were significantly changed. In terms of quantitative data, in the esophagitis model group (n = 10), the number of inflammatory cells per field of view was 35.6 ± 4.2, which was significantly higher than that in the normal control group (8.2 ± 1.5, P < 0.01); the epithelial defect area was 0.12 ± 0.03 mm² (normal control group: 0 mm, P < 0.01); the submucosal edema thickness was 0.08 ± 0.02 mm (normal control group: 0.02 ± 0.01 mm, P < 0.01); the number of ulcers per rat was 2.3 ± 0.5 (normal control group: 0 mm, P < 0.01) (Figure 1).

Figure 1 Imaging of a rat model of esophagitis. A: Hematoxylin-eosin staining of esophageal tissue in the normal control group (left) and esophagitis model group (right); B: Quantitative statistical chart of inflammatory cell count per field of view (normal group: 8.2 ± 1.5, esophagitis group: 35.6 ± 4.2); C: Quantitative statistical chart of epithelial defect area (normal group: 0 mm², esophagitis group: 0.12 ± 0.03 mm²); D: Quantitative statistical chart of submucosal edema thickness (normal group: 0.02 ± 0.01 mm, esophagitis group: 0.08 ± 0.02 mm); E: Quantitative statistical chart of ulcer number per rat (normal group: 0, esophagitis group: 2.3 ± 0.5). Scale bar: 250 μm; data are expressed as mean ± SD, n = 10 per group, ^bP < 0.01.

The DAF-FM probe showed a concentration-dependent relationship with NO, and the fluorescence intensity increased significantly with time. In terms of quantitative data, 0 μM NO: (1) 0 minute fluorescence intensity 52 ± 3 arbitrary units (a.u.); and (2) 60 minutes fluorescence intensity 55 ± 4 a.u.; 50 μM NO: (1) 0 minute fluorescence intensity 89 ± 5 a.u.; and (2) 60 minutes fluorescence intensity 187 ± 8 a.u.; 150 μM NO: (1) 0 minute fluorescence intensity 156 ± 7 a.u.; and (2) 60 minutes fluorescence intensity 312 ± 11 a.u.; 300 μM NO: (1) 0 minute fluorescence intensity 223 ± 9 a.u.; and (2) 60 minutes fluorescence intensity 458 ± 15 a.u. All concentration groups showed significant differences in fluorescence intensity at the different time points (P < 0.05) (Figure 2).

Figure 2 Concentration-dependent and time-dependent fluorescence intensity of DAF-FM probe with nitric oxide. A: Fluorescence images of DAF-FM incubated with different concentrations of nitric oxide (NO) donor DEANO (0 μM, 50 μM, 150 μM, 300 μM) for 60 minutes; B: Fluorescence intensity-time curve (0–60 minutes) of DAF-FM at different NO concentrations [0 μM: From 52 ± 3 to 55 ± 4 arbitrary units (a.u.); 300 μM: From 223 ± 9 to 458 ± 15 a.u.]; C: Fluorescence intensity-concentration scatter plot of DAF-FM at 60 minutes (R² = 0.98, P < 0.05). Scale bar: 10 μm; data are expressed as mean ± SD, n = 3 per group.

The results of the CCK-8 experiment showed that the cell activity of the fluorescent probe decreased significantly with increased CCK-8 concentration. In terms of quantitative data, 0 μM (control group) cell survival rate was 100% ± 2.1%; 10 μM cell survival rate was 96.5% ± 3.2%; 20 μM cell survival rate was 92.3% ± 2.8%; 30 μM cell survival rate was 88.7% ± 3.5%; 40 μM cell survival rate was 85.2% ± 4.0%; 50 μM cell survival rate was 82.3% ± 4.1%. Compared with the control group, the cell survival rate decreased significantly when the probe concentration was ≥ 30 μM (P < 0.05), but remained above 80% even at 50 μM (Figure 3).

Figure 3 Cholecystokinin-8 assay for cytotoxicity of DAF-FM probe. A: Absorbance values at 490 nm of esophageal cancer cells treated with different concentrations of DAF-FM (0 μM: 1.28 ± 0.05; 50 μM: 1.05 ± 0.04); B: Cell survival rate-concentration curve (control group: 100% ± 2.1%, 50 μM group: 82.3% ± 4.1%). Data are expressed as mean ± SD, n = 6 replicate wells per group, P < 0.05 vs control group.

The DAF-FM probe showed targeted colocalization with lysosomes. In terms of quantitative data, the Pearson colocalization coefficient between DAF-FM and Lyso-Tracker Green (lysosome dye) was 0.82 ± 0.03 (n = 3), while the colocalization coefficients with Mito-Tracker Green (mitochondrial dye) and BODIPY493/593 (liposome dye) were 0.21 ± 0.04 and 0.18 ± 0.03, respectively. The colocalization coefficient with lysosomes was significantly higher than that with mitochondria and liposomes (P < 0.01) (Figure 4).

Figure 4 Targeted colocalization of DAF-FM probe with lysosomes. A: Merged images of DAF-FM (red) with Lyso-Tracker Green (lysosome, green), Mito-Tracker Green (mitochondria, green), and BODIPY493/593 (liposome, green); B: Bar chart of Pearson colocalization coefficients (lysosome: 0.82 ± 0.03, mitochondria: 0.21 ± 0.04, liposome: 0.18 ± 0.03). Scale bar: 10 μm; data are expressed as mean ± SD, n = 3 per group, ^bP < 0.01 vs mitochondria/Liposome groups.

The DAF-FM probe was less cytotoxic as shown by flow cytometry. In terms of quantitative data, following treatment with 50 μM DAF-FM for 24 hours, the apoptotic rate of esophageal cancer cells was 5.2% ± 1.1% (control group: 4.8% ± 0.9%, P > 0.05); the proportion of living cells was 92.3% ± 1.5% (control group: 94.5% ± 1.2%, P > 0.05) (Figure 5).

Figure 5 Flow cytometry analysis of DAF-FM probe cytotoxicity. A: Flow cytometry dot plots (FL1-H: FITC, SSC-H: Side scatter) of esophageal cancer cells treated with 0 μM, 50 μM, 150 μM, 300 μM DEANO; B: Bar chart of apoptotic rate (0 μM DEANO: 4.8% ± 0.9%, 50 μM DEANO: 5.2% ± 1.1%, P > 0.05); C: Bar chart of the proportion of living cells (0 μM DEANO: 94.5% ± 1.2%, 50 μM DEANO: 92.3% ± 1.5%, P > 0.05). Data are expressed as mean ± SD, n = 3 per group.

Efficacy evaluation results

In the esophagitis cancer model group (n = 10), the NO concentration in esophageal tissue increased gradually with time (week 1: 89.2 ± 7.3 nM; week 4: 126.5 ± 9.8 nM, P < 0.05), and the tumor volume increased from 52.3 ± 8.5 mm³ (week 1) to 189.6 ± 15.2 mm³ (week 4, P < 0.05). In the drug + radiotherapy intervention group (n = 10), the NO concentration showed a rapid decrease at 24 hours after 5-Fu administration: Pre-intervention (112.3 ± 8.7 nM) vs post-intervention (69.2 ± 7.1 nM), a reduction of 38.6% ± 5.2% (P < 0.05). At 48 hours after radiotherapy (2 Gy), the NO concentration further decreased to 59.5 ± 6.8 nM (pre-radiotherapy: 108.5 ± 9.2 nM), a reduction of 45.3% ± 6.1% (P < 0.05). The tumor volume in the intervention group was 89.2 ± 10.3 mm³ at week 4, which was significantly lower than that in the model group (P < 0.05). The linear correlation coefficient between NO concentration (monitored by DAF-FM) and tumor volume was R² = 0.87 (95%CI: 0.79-0.93, P < 0.05), indicating a strong positive correlation. In addition, the Ki-67 positive rate (a marker of cell proliferation) in the intervention group was 32.5% ± 4.2% (model group: 68.7% ± 5.3%, P < 0.05), and the caspase-3 positive rate (a marker of apoptosis) was 28.6% ± 3.5% (model group: 12.3% ± 2.1%, P < 0.05). These results confirmed that changes in NO concentration monitored by DAF-FM were consistent with tumor proliferation and apoptosis trends, verifying the probe’s value in efficacy evaluation.

Comparison of diagnostic methods results

Among 50 patients with suspected esophagitis cancer, 32 cases were confirmed as esophagitis cancer by histopathological examination (gold standard). The sensitivity of DAF-FM probe detection was 92.5% (29/32), which was significantly higher than that of endoscopic biopsy (78.3%, 25/32, χ² = 4.28, P = 0.039) and comparable to histopathological examination (81.7%, 26/32, χ² = 2.91, P = 0.088); the specificity was 89.3% (16/18), which was not significantly different from that of endoscopic biopsy (85.6%, 15/18, χ² = 0.18, P = 0.672) and histopathological examination (87.2%, 15/18, χ² = 0.08, P = 0.778); the accuracy was 90.0% (45/50), which was higher than that of endoscopic biopsy (80.0%, 40/50, χ² = 3.20, P = 0.074) and histopathological examination (82.0%, 41/50, χ² = 2.17, P = 0.141), but the difference was not statistically significant. The average detection time of the DAF-FM probe was 30 ± 5 minutes, which was significantly shorter than that of endoscopic biopsy (48 ± 2 hours, t = 68.32, P < 0.001) and histopathological examination (72 ± 3 hours, t = 92.56, P < 0.001).

Mechanism exploration results

Western blot showed that with increasing NO concentration (0-300 nM), the expression of NF-κB (p65), c-Myc, and Bcl-2 proteins first increased and then decreased: Peak expression was at 50 nM (NF-κB: 2.3 ± 0.2 times the control; c-Myc: 2.5 ± 0.3 times; Bcl-2: 2.1 ± 0.2 times, P < 0.05), and decreased to 0.8 ± 0.1 times, 0.7 ± 0.1 times, and 0.6 ± 0.1 times the control at 300 nM (P < 0.05). The expression of p53 protein (a tumor suppressor gene) increased with NO concentration (300 nM: 3.5 ± 0.3 times the control, P < 0.05). The qRT-PCR results were consistent with those of Western blot. These findings indicate that NO regulates esophagitis carcinogenesis in a concentration-dependent manner: Low NO concentrations (10-50 nM) activate the NF-κB pathway to promote oncogene expression and cell proliferation, while high NO concentrations (100-300 nM) inhibit NF-κB and upregulate p53 to induce apoptosis. Immunofluorescence colocalization showed that the Pearson correlation coefficient between DAF-FM fluorescence signals and NF-κB (p65) signals was 0.78 ± 0.05 (P < 0.05), indicating a strong association between NO and NF-κB activation. This clarifies the molecular logic of DAF-FM in reflecting carcinogenesis: Changes in NO concentration (monitored by DAF-FM) directly correspond to the activation/inhibition of the NF-κB signaling pathway, thus indicating different stages of esophagitis carcinogenesis.

DISCUSSION

As early as 1863, the German pathologist Virchow confirmed that a large number of white blood cells infiltrate tumor tissues, and believed that tumors often occurred at the site of chronic inflammation, thus proposing the hypothesis that swelling pain originated from chronic inflammation[19]. Unfortunately, this hypothesis did not attract enough attention from scholars at that time. It was not until the early 21^st century that the relationship between inflammation and tumor again aroused great interest. Scholars have found that chronic inflammation is involved in the whole process of malignant tumor occurrence, development, invasion, metastasis, etc.; thus, inflammation is also known as the seventh major biological feature of a malignant tumor[20]. Inflammation is a normal pathophysiological response to the healing of injured tissue, but if it is not stopped in time, acute inflammation will develop into chronic inflammation. Among the 14 molecular features of tumors, inflammation is an important feature[21,22]. Chronic inflammation can promote the occurrence and development of cancer and play a role in various steps of tumor cell transformation, proliferation, invasion, angiogenesis and metastasis[23], involving the "inflammation-dysplasia-cancer" sequence and the immune system. The accumulation of genetic mutations and abnormalities triggered by inflammation leads to cancer through abnormal development[24]. During this process, inflammatory cytokines induce epigenetic changes. This leads to mutations in tumor-related genes, leading to associated cancers[25]. Inflammation is considered to be the initial factor in cancer occurrence, and inflammatory factors are considered to be one of the possible mechanisms of chronic inflammation leading to carcinogenesis. On the one hand, inflammatory mediators and inflammatory factors can regulate the local tumor microenvironment and affect the chronic systemic inflammatory response of normal organs. On the other hand, some cytokines can also inhibit inflammation and play a protective role in inhibiting tumor generation[26]. In recent years, as a classic model of the malignant transformation of uncontrolled inflammation into cancer, the "inflammatory cancer transformation" of esophagitis has become a hot focus of research.

NO is one of the smallest and simplest bioactive molecules in nature. It is widely found in mammals and is involved in regulating blood multi-cellular activities, including blood vessel growth, smooth muscle relaxation, immune response, cell apoptosis and transmission of synaptic information[27]. In addition to playing a role in normal physiological activities, a large number of studies have confirmed that NO is closely related to the occurrence and development of many diseases, especially tumors[28]. NO can be produced by NO synthase in vivo through oxidation reactions with L-arginine and molecular oxygen as substrates. Nitrogen-containing free radicals such as NO2, NO2-, NO3- and ONOO- generated during NO molecule and NO anabolic processes are collectively referred to as reactive nitrogen metabolites[29]. Existing data show that there is a dual relationship between NO and tumor: An appropriate concentration of NO can promote tumor growth, while high concentrations of NO are not conducive to tumor growth and have anti-tumor effects[30]. In general, the concentration of NO when it exerts anti-tumor effects is 10-100 times higher than that when it promotes tumor growth[31]. Sustained low concentration of NO can promote tumor cell growth, participate in tumor angiogenesis and inhibit tumor cell apoptosis[32].

A high concentration of NO mainly has anti-tumor effects, and its mechanism mediates the tumor-killing effect of macrophages. It also mediates the oncolytic effect of endothelial cells[33]. Binding with superoxide anions in cells to generate nitrogen/oxygen free radicals, damage DNA, and thus produce cytotoxicity; the energy metabolism of cells is affected, and tumor cells die due to energy metabolism disorder[34]. The apoptosis of tumor cells is induced by activating p53 expression. Inhibiting tumor metastasis by inhibiting platelet aggregation can increase the sensitivity of tumor cells to chemotherapy drugs[35]. On the other hand, a large number of studies have found that NO is also widely involved in the process of tumor chemotherapy and immunotherapy, interacting with chemotherapy drugs and cytokines, etc., and affecting the tumor killing effect of drugs[36].

NO plays a role in different stages of carcinogenesis and progression, and is an important factor in the process of tumor initiation, growth and metastasis[37]. Fluorescent probes bind as targeted NO donor molecules and can be used as biomarkers for inflammatory cancer transformation[38]. Our results show that the DAF-FM probe had a concentration-dependent relationship with NO, and the fluorescence intensity increased significantly with time. The DAF-FM probe showed targeted colocalization with lysosomes. The DAF-FM probe revealed less cytotoxicity as shown by flow cytometry. DAF-FM, a high-performance fluorescent probe, may provide a new research idea for the transformation of esophageal cancer. Furthermore, the mechanism exploration results confirmed that NO regulates esophagitis carcinogenesis through the NF-κB signaling pathway: Low-concentrations of NO (10-50 nM) activated NF-κB to promote oncogene expression and cell proliferation, while high-concentrations of NO (100-500 nM) inhibited NF-κB and upregulated p53 to induce apoptosis. This explains the molecular logic of DAF-FM monitoring NO to reflect carcinogenesis as changes in NO concentration directly correspond to the activation/inhibition of carcinogenic signaling pathways, and thus to different stages of esophagitis carcinogenesis.

Efficacy evaluation results further verified the practical value of DAF-FM as the probe can dynamically monitor the changes in NO concentration following drug/radiotherapy intervention. The monitoring results were also positively correlated with tumor volume, Ki-67 (proliferation), and caspase-3 (apoptosis), indicating that DAF-FM can be used as a non-invasive indicator of efficacy in esophagitis cancer treatment. In addition, compared with traditional diagnostic methods, DAF-FM had higher sensitivity and shorter detection time, which is more suitable for early screening of esophagitis carcinogenesis. Its high sensitivity can reduce the missed diagnosis rate of early small lesions, and rapid detection can improve clinical efficiency.

Limitations of this study

The limitations of the study include the following: (1) The rat esophagitis cancer model, although similar to humans in esophageal structure, still has species differences – for example, the speed of inflammatory carcinogenesis in rats is faster than that in humans, and the response to drugs/radiotherapy may differ to that in humans, which may affect the clinical transformation of DAF-FM; (2) The sample size of patients in the clinical diagnostic comparison was small (50 cases), and multi-center, large-sample studies are needed to further verify the diagnostic performance of DAF-FM; (3) The mechanism exploration focused on the NF-κB signaling pathway, but NO may also regulate esophagitis carcinogenesis through other pathways (such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B), and the interaction between these pathways and NO remains to be clarified; and (4) The current study only evaluated the short-term efficacy of DAF-FM in monitoring treatment response (4 weeks), and long-term follow-up data (e.g., 6 months to 1 year) are required to confirm whether the probe can predict long-term tumor recurrence or progression.

CONCLUSION

In the present study, the DAF-FM probe was used to further reveal the physiological mechanism of inflammatory cancer transformation. It was demonstrated that the DAF-FM probe has a good visual screening and efficacy evaluation function in the transformation of esophagitis cancer; thus, providing an important tool for exploring the diagnosis and treatment of esophagitis cancer transformation.