Sprayable fluorescent probe lights up tiny ovarian cancer lesions in the operating room

Ovarian cancer remains the deadliest malignancy of the female reproductive system, with a five-year survival rate that has stubbornly remained below 44 percent for more than two decades. Roughly 70 percent of patients are already at an advanced stage when they are first diagnosed, and the single most important determinant of their survival is whether surgeons can remove every visible tumor during cytoreductive surgery. Yet the tools available to locate these lesions before and during an operation are strikingly limited. CT and MRI scans, along with blood markers such as CA125 and HE4, correlate with tumor burden but cannot pinpoint diffuse seedings on the peritoneum or mesentery. Laparoscopy helps but adds cost, and the detection of occult implants still depends largely on the surgeon’s eyes and fingers, a subjective process prone to error. Against this backdrop, a team of clinicians and chemists in China has now reported a promising step toward making hidden ovarian cancer lesions glow in plain sight.

In a pilot study published in Holistic Integrative Oncology, researchers from Liaoning Cancer Hospital and Dalian University of Technology evaluated a sprayable fluorescent probe that targets gamma-glutamyl transpeptidase, an enzyme abbreviated γ-GGT or GGT2. The enzyme sits on the membranes of mammalian cells and is normally abundant in secretory organs such as the liver, kidney, and pancreas, but its expression and activity rise sharply in many malignancies, including liver, lung, colorectal, breast, and ovarian cancers. Previous work has suggested that γ-GGT helps tumors progress, invade, and resist chemotherapy by reshaping intracellular redox metabolism, and elevated pretreatment serum levels of the enzyme have been linked to advanced stage and poor prognosis in epithelial ovarian cancer. That combination of high tumor expression and clinical relevance made γ-GGT an attractive molecular bullseye for intraoperative imaging.

The probe itself, designated NIR-SN-GGT, is a product of a medical-engineering collaboration between the cancer hospital and the State Key Laboratory of Fine Chemicals in Dalian. Chemically, it couples a dicyanoisophorone-based near-infrared fluorophore to a γ-glutamyl recognition unit through an amide bond. In its intact state the molecule is essentially dark: the γ-glutamyl group acts as an electron-withdrawing quencher that suppresses intramolecular charge transfer, the process responsible for fluorescence. When the probe encounters γ-GGT on the surface of a cancer cell, the enzyme cleaves the γ-glutamyl bond, releasing a free amine that restores charge transfer and triggers a rapid turn-on fluorescence signal in the near-infrared region, with an emission maximum around 650 nanometers, excitation near 445 nanometers, and a large Stokes shift of roughly 205 nanometers. Because the reaction occurs only where the enzyme is abundant, background fluorescence in healthy tissue stays negligible, which is precisely what a surgeon needs to distinguish tumor from normal tissue in real time.

Crucially, the team did not stop at cell cultures or mouse models, the point at which most targeted fluorescent probes have historically stalled. Instead, they built a three-tier validation pipeline that moved from bioinformatics to organoids, then to frozen tissue sections, and finally to fresh human specimens removed during surgery. Analysis of The Cancer Genome Atlas confirmed elevated GGT2 mRNA in ovarian tumors compared with normal ovary, and immunohistochemistry on archival paraffin blocks from 36 high-grade serous ovarian cancer cases showed robust γ-GGT protein on cancer cell membranes. The researchers then grew patient-derived ovarian cancer organoids in three-dimensional matrix cultures and observed distinct probe fluorescence within these miniature tumors, confirming that the probe can label γ-GGT-positive cells in a system that mimics the tumor microenvironment far better than flat cell monolayers.

The next tier involved frozen sections cut at 100-micrometer thickness from freshly resected cancer and pericancerous tissue. After spraying the sections with a 10 micromolar probe solution and waiting just one minute, confocal microscopy revealed a fluorescence pattern that colocalized almost perfectly with DAB immunostaining for γ-GGT, with markedly stronger signal in cancerous regions than in adjacent non-cancerous tissue. This spatial agreement between enzyme activity imaging and classical pathology provided the bridge from in vitro validation to real human tissue, setting the stage for the ex vivo experiments that form the heart of the study.

Between February and November 2023, the team collected fresh tissue from 16 patients aged 46 to 77 who underwent primary, interval, or secondary cytoreductive surgery for epithelial ovarian cancer, most of them at advanced FIGO stage III or IV. From these patients they harvested 42 confirmed cancerous lesions, including 18 primary ovarian lesions, 15 omental metastases, and 9 peritoneal metastases, spanning diameters of 0.5 centimeters, 0.3 centimeters, and smaller than 0.3 centimeters. Each specimen was sprayed with probe solution at 1, 5, or 10 micromolar within 30 to 40 minutes of resection, then imaged every minute for half an hour and again at one hour and five hours using a simple setup: an iPhone 12 mini camera fitted with an emission filter, working at a distance of 15 to 20 centimeters under dark conditions. Signal-to-background ratios, calculated as average lesion fluorescence divided by average fluorescence of surrounding normal tissue, served as the primary metric, with values above 1.2 considered visible to the naked eye.

The optimization experiments delivered clear answers. Across all 42 lesions, every sample produced a signal-to-background ratio above 1, yielding 100 percent fluorescence detection, and 95.2 percent were visible without any image processing. The 10 micromolar concentration proved to be the minimum effective dose, achieving signal-to-background ratios of 1.40 plus or minus 0.17 for primary lesions and 1.32 plus or minus 0.12 for omental metastases, significantly better than the 1.17 and 1.27 values seen at lower concentrations. Just as importantly, the imaging window proved remarkably forgiving: fluorescence appeared within one minute of spraying and remained stable from one minute to one hour, with ratios hovering between 1.26 and 1.29, before fading by five hours. Lesion size made no difference, with sub-0.3-centimeter implants imaged just as brightly as larger ones, a finding with obvious implications for catching the millimeter-scale seedings that surgeons most often miss.

The clinical validation phase then put the optimized protocol to the test on 27 sub-0.3-centimeter lesions from 8 patients, along with adjacent normal tissues and a negative control of normal peritoneum. Fluorescence imaging identified 26 of the 27 cancerous lesions, a true-positive rate of 96.3 percent, and 23 lesions were visible to the naked eye, a macroscopic detection rate of 81.5 percent. Only one false positive appeared, in a sample of normal peritoneal tissue that pathology confirmed lacked γ-GGT expression; the spurious signal was attributed to residual red blood cells on the tissue surface, whose protoporphyrin content autofluoresces in the same spectral region. Immunohistochemistry confirmed membrane γ-GGT expression in 100 percent of cancerous lesions in both cohorts while normal tissues stained negative, underscoring the target’s specificity. The authors note that meticulous intraoperative hemostasis and thorough saline rinsing of tissue surfaces before spraying are essential precautions to keep blood-related false positives in check.

Statistical analysis of the clinical variables added a note of caution for future use. Univariate analysis found that preoperative serum γ-GGT levels and treatment modality both influenced imaging quality, with patients whose serum levels reached at least 20 units per liter showing higher signal-to-background ratios. In multivariate analysis, only the preoperative treatment approach remained independently predictive, most likely because neoadjuvant chemotherapy can reduce γ-GGT expression or activity in tumor tissue and thereby weaken probe binding. Encouragingly, clinical stage, lesion location, and pathological type did not alter probe performance, suggesting the technique is robust across the diverse clinical presentations of ovarian cancer.

The authors are candid about the study’s limits. The cohort of 16 patients is too small for firm statistical conclusions, and larger trials will be needed to stratify effects of specific chemotherapy regimens and timing of interval surgery. The single false negative occurred in a γ-GGT-positive lesion buried more than three millimeters beneath the tissue surface, a reminder that near-infrared light at 650 nanometers penetrates tissue only shallowly; deeper lesions may require surgical exposure or next-generation probes emitting in the second near-infrared window above 1000 nanometers. Standardized fluorescence evaluation criteria will also be needed to reduce observer variability. Even so, the head-to-head comparison is striking: the FDA-approved folate receptor-alpha probe pafolacianine detects ovarian cancer lesions with 83.0 percent sensitivity and a 32.7 percent false-positive rate, whereas the sprayed γ-GGT probe achieved a 98.6 percent true-positive rate with only a 1.4 percent false-positive rate in the ex vivo experiments, while topical delivery slashes the dose required compared with intravenous agents and reduces the risk of systemic toxicity. If larger clinical trials confirm these results, a bottle of enzyme-activated dye and a filtered smartphone camera could one day become standard equipment in the fight against one of medicine’s most elusive cancers.

Subject of Research: Sprayable γ-GGT-activated fluorescent probe for intraoperative visualization of epithelial ovarian cancer

Article Title: Exploration of the application of the sprayed γ-GGT fluorescent probe for visual imaging of epithelial ovarian cancer: a pilot study

Article References: Liu, Z., Zhang, M., Li, H., Liu, L., Chen, F., Li, M., Gao, Y., Wang, D., & Yang, Z. (2026). Exploration of the application of the sprayed γ-GGT fluorescent probe for visual imaging of epithelial ovarian cancer: a pilot study. Holistic Integrative Oncology, 5(1), Article 46. https://doi.org/10.1007/s44178-026-00268-7

Image Credits: AI Generated

DOI: 10.1007/s44178-026-00268-7

Keywords: ovarian cancer, fluorescent probe, gamma-glutamyl transpeptidase, fluorescence imaging, intraoperative imaging, cytoreductive surgery, organoids, signal-to-background ratio, near-infrared fluorescence, tumor imaging, pafolacianine, precision oncology