The Critical Need for Precision in Cancer Surgery

In Japan, cancer remains a leading cause of death, with approximately 979,300 new cases estimated in 2024 alone, including high incidences of breast, colorectal, and ovarian cancers where surgery plays a pivotal role. Successful outcomes depend on surgeons' ability to excise all tumor tissue while sparing healthy organs. However, microscopic remnants or millimeter-sized lesions often escape detection, leading to recurrence rates as high as 20-30% in some peritoneal dissemination cases. Fluorescence-guided surgery (FGS) has emerged as a promising solution, using near-infrared or visible light probes to highlight tumors intraoperatively. Yet, traditional probes suffer from high background fluorescence from non-specific activation, obscuring tiny lesions.

Researchers at the University of Tokyo (UTokyo) have addressed this challenge head-on with a groundbreaking bioorthogonal fluorescence probe system, enabling unprecedented low-background, high-contrast tumor imaging. This innovation from UTokyo's Graduate School of Medicine promises to transform targeted tumor imaging for cancer surgery, particularly for HER2-positive tumors prevalent in breast and ovarian cancers.

Bioorthogonal Chemistry: The Foundation of Selective Imaging

Bioorthogonal chemistry refers to chemical reactions that occur within living systems without interfering with native biological processes. First conceptualized by Carolyn Bertozzi, it relies on non-natural functional groups like tetrazines or cyclooctynes that react selectively. In cancer imaging, bioorthogonal probes remain inert until triggered by a pre-delivered targeting agent, minimizing off-target activation.

UTokyo's approach builds on this by pairing a quenched fluorescent dye with an engineered enzyme. The probe, HMRef-β-D-fucose, is a rhodol derivative where the β-D-fucose sugar quenches fluorescence via spirocyclization. Only the specific glycosidase enzyme cleaves the sugar, releasing the bright HMRef fluorophore. This bioorthogonal pairing ensures the probe ignores endogenous mammalian enzymes, unlike galactose-based probes activated by beta-galactosidase naturally present in tissues.

This design achieves signal-to-background ratios (SBR) far superior to conventional systems, critical for visualizing sub-millimeter tumors during surgery.

Engineering a High-Performance Reporter Enzyme

The magic lies in the reporter enzyme: Td2F2, a metagenomic glycosidase from GH1 family discovered in compost microbes. Wild-type Td2F2 had modest activity on the probe (k_cat/K_m low), so UTokyo researchers employed directed evolution. Through fluorescence-activated cell sorting (FACS) screening using a trapping variant (SPiDER-β-D-fucose), they generated Mutant A (E27G, I34T, F243L, E296G). This variant boasts a 7.3-fold higher catalytic efficiency (k_cat/K_m = 3.3 × 10^5 M⁻¹ s⁻¹), rivaling beta-galactosidase on its substrate.

Quantum mechanics/molecular mechanics (QM/MM) simulations revealed E296G disrupts a salt bridge, widening the active site for better probe access. The monomeric enzyme was fused genetically to anti-HER2 nanobody (2Rs15d) or conjugated via maleimide to trastuzumab or avidin-biotin, ensuring tumor-specific delivery.

Step-by-Step: From Injection to Intraoperative Glow

1. Tumor Targeting: Engineer enzyme conjugate (e.g., Td2F2-Mutant A fused to HER2 affibody) injected intravenously 24-48 hours pre-surgery, binds selectively to HER2-overexpressing cancer cells.
2. Probe Administration: Bioorthogonal probe HMRef-β-D-fucose injected intraperitoneally shortly before imaging; remains dark due to quenching.
3. Enzymatic Activation: Tumor-bound enzyme hydrolyzes glycosidic bond, releasing fluorescent HMRef and generating quinone methide for intracellular trapping (in SPiDER variant).
4. Intraoperative Imaging: Near-infrared excitation reveals glowing tumors with minimal background; surgeons excise precisely using standard FGS cameras.

In mouse models, this yielded vivid millimeter-sized lesion visualization in peritoneal cavities.

Triumph in Mouse Models: Low Background, High Contrast

Tested on HER2-positive SKOV-3 ovarian cancer peritoneal dissemination in nude mice, the system lit up tumors with SBR significantly higher than beta-gal controls (p < 0.05). Background in intestines, uterus negligible, unlike controls showing autofluorescence. Mesentery dissection revealed cancer nodules clearly. Live-cell imaging confirmed specificity post-wash. Mutant A outperformed wild-type, matching beta-gal signal without drawbacks.

These results highlight potential for HER2+ cancers, affecting ~15-20% of breast cancers in Japan.

Superiority Over Existing FGS Technologies

• Low Background: Bioorthogonal design evades endogenous enzymes; beta-gal shows 10x higher off-target.
• Tumor Specificity: Modular targeting (affibody, antibodies) adaptable to antigens like EGFR, PSMA.
• Brightness: Engineered enzyme efficiency ensures strong signal in vivo.
• Safety: Non-toxic probe/enzyme; no immunogenicity in short-term mouse studies.

Compared to ICG or antibody-dye conjugates used in Japan, this offers >10-fold SBR improvement for micro-lesions.

The Visionaries: UTokyo's Research Team

Led by Associate Professor Ryosuke Kojima, an expert in chemical biology and molecular imaging at UTokyo's Graduate School of Medicine. His lab focuses on fluorogenic probes for disease detection. Co-senior author Professor Yasuteru Urano pioneers activatable probes. Collaborators from Kyoto University contributed enzyme engineering. Kojima notes, “This allowed us to see tiny, millimeter-sized tumor lesions with extremely low background noise, a level of contrast that could be very useful during surgery.”

UTokyo's prowess in interdisciplinary research, blending chemistry, biology, and engineering, drives such innovations. For aspiring researchers, opportunities abound in research jobs at leading Japanese universities.

Transforming Cancer Care in Japan

With ~393,100 cancer deaths projected for 2024, precise surgery is vital. Colorectal (15-16% incidence), breast (22% female), and ovarian cancers often require FGS. Japan's aging population amplifies demand, yet surgeon shortages loom. This probe could reduce recurrences, improve 5-year survival (already high at 60-70% for many cancers), and cut healthcare costs. Initial research tool use may precede clinical trials.

Read UTokyo's full press release for visuals.

Global Impact and FGS Evolution

Bioorthogonal FGS aligns with Japan's indocyanine green (ICG) adoption trends, potentially integrable with existing systems. Adaptable for drug delivery (prodrug activation). Challenges: immunogenicity mitigation, human trials. UTokyo's work positions Japan as a leader in molecular imaging.

Future Horizons: Clinical Translation Roadmap

Next: pharmacokinetics optimization, multi-antigen targeting, Phase I trials. Partnerships with pharma like higher-ed jobs in drug development. Long-term: standard in robotic surgery suites.

UTokyo's Enduring Legacy in Cancer Research

UTokyo's labs, including Cancer Biology and Immunotherapeutics, contribute globally. This probe exemplifies translational excellence. Explore university jobs in Japan or research positions to join such frontiers.

Career Insights and Next Steps

For higher ed professionals, this underscores demand for chemical biologists. Check career advice, rate professors, or browse higher ed jobs. Stay ahead with university jobs alerts.

Photo by Craig Cameron on Unsplash