Colorectal cancer (CRC) is a major global health challenge. Surgical resection remains the cornerstone of treatment; however, microscopic residual lesions after surgery frequently lead to recurrence, while conventional adjuvant chemotherapy and radiotherapy cause severe systemic toxicity that patients can hardly tolerate. To address this core dilemma, we developed RectomeDy FotoZymogen, a red-light–induced, locally targeted bacterial therapy system designed for precise and controllable postoperative treatment.

Figure 1：Conceptual Schematic of the RectomeDy FotoZymogen Therapy.

RectomeDy FotoZymogen integrates four core functional modules, enabling intelligent closed-loop control over the entire therapeutic system:（1）Precise Targeting and Anchoring:The INP-HlpA system adheres to HSPG on CRC cells, resisting bowel peristalsis to ensure high local drug concentration.（2）Red Light Inducible Expression:The NETMAP switch utilizes NIR's high penetration for rapid, precise activation and quantitative dosage control of therapeutic gene expression.（3）Dual-Payload Collaborative Therapy：Co-expression of Coagulase (Coa) (physical obstruction) and the PD-L1 nanobody (PD-L1 nb) (immune activation) realizes a synergistic therapeutic effect.（4）High-Redundancy Conditional Suicide:A multi-logic-gated self-destruct system, named the ORION Kill-Switch, utilizes the MazF/MazE toxin-antitoxin system for its construction.

Figure 2：Four Core Functional Modules of RectomeDy FotoZymogen. The system comprises: (1) an adhesion system mediated by INP-HlpA; (2) a responsive expression system mediated by the NETMAP red light promoter; (3) a therapeutic system mediated by Coagulase (Coa) and the PD-L1 nanobody (PD-L1 nb); and (4) a triple safety system with an "OR" gate logic composed of arabinose and red light.

Delivered through a rectal suppository and activated by localized illumination, the system confines therapeutic effects strictly to postoperative sites, eliminating systemic toxicity. With its built-in biosafety, spatiotemporal precision, and clinical affordability, RectomeDy FotoZymogen provides a novel, safe, and effective strategy for preventing CRC recurrence after surgery.

Figure 3：Advantages of the RectomeDy FotoZymogen System for Local Postoperative Therapy：Bio-Safety ,Spatio-Temporal Precision and Clinical Affordabilty.

Our inspiration originates from the personal experience of our beloved teacher, Mr. X, known for his vivid and passionate teaching style that made every class inspiring and full of life. One year ago, he was diagnosed with colorectal cancer and courageously underwent abdominoperineal resection, a life-saving but physically and emotionally demanding surgery that required a permanent colostomy due to the tumor’s location. While he survived, he told us that his greatest fear was not the stoma itself, but the invisible residual cancer cells that might cause recurrence. To combat this 30% recurrence risk [1], he endured months of adjuvant chemotherapy, which brought numbness, nausea, and vomiting [2], eventually forcing him to leave the job he loved.

Figure 4: Inspired by the painful experience of Mr. X, we designed RectomeDy FotoZymogen—a red-light–controlled, site-specific engineered bacterial therapy system for safer and smarter post-surgical adjuvant cancer care.

His painful experience revealed a harsh reality — there is no low-toxicity adjuvant therapy capable of precisely eliminating postoperative minimal residual disease [3]. It was his story that inspired us to focus on post-surgical recurrence prevention while avoiding systemic toxicity entirely. Guided by this motivation, we designed RectomeDy FotoZymogen, a red-light–controlled, site-specific engineered bacterial therapy system that represents a step toward safer, smarter, and more compassionate cancer care [4].

Colorectal cancer (CRC) is one of the most prevalent and lethal malignancies worldwide. According to the WHO GLOBOCAN database, CRC ranks as the third most common cancer among men and the second among women, with approximately 1.8 million new cases and 881,000 deaths globally in 2018 [5]. Because early-stage CRC often presents with nonspecific or even absent symptoms, coupled with limited screening coverage, a large proportion of patients are diagnosed at advanced stages, when curative options are limited [6]. Advanced CRC is characterized by high invasiveness and strong metastatic potential, resulting in a significantly reduced 5-year survival rate compared to early-stage disease [7]. Although surgical resection remains the cornerstone of curative treatment, the success of surgery often marks the beginning, rather than the end, of the therapeutic journey. Clinical evidence indicates that even after complete resection of the primary tumor, minimal residual disease (MRD) or micrometastases may persist, leading to local recurrence or distant metastasis [8]. The recurrence rate can reach nearly 30%, making postoperative adjuvant therapy indispensable for long-term disease control [9].

Figure 5: Global statistics illustrating the high incidence and mortality of colorectal cancer, highlighting its severe impact on public health.

However, current adjuvant therapies exhibit significant limitations in efficacy, safety, and tolerability. Chemotherapy, radiotherapy, and emerging targeted or immunotherapies have improved survival outcomes, yet each carries substantial drawbacks.

Table 1: Comparison of current adjuvant therapies for colorectal cancer, summarizing their mechanisms, advantages, and major limitations.

Figure 6: Worldwide colorectal cancer incidence and mortality rates (age adjusted according to the world standard population, per 100 000) in males in 2012 (GLOBOCAN 2012).

Additionally, surgical procedures for CRC often result in anastomotic sites or stomas, which are fragile and microbially complex, creating additional challenges for postoperative drug delivery and local targeting. For many patients, long-term chemotherapy causes pain, nausea, vomiting, alopecia, and cumulative neurotoxicity, severely impairing quality of life and treatment compliance [10]. Radiotherapy, although locally precise, can injure healthy intestinal epithelium in the pelvic region, leading to ulceration, chronic inflammation, and diarrhea [11]. Targeted and immune therapies, while molecularly advanced, remain financially prohibitive and clinically selective, preventing widespread adoption [12].

Figure 7: Overview of current cancer therapies and their side effects, highlighting the limitations of conventional chemotherapy and radiotherapy.

Given these realities, there remains a pressing unmet need for a new generation of postoperative adjuvant therapies—ones that are localized, controllable, and safe, capable of complementing surgery without adding systemic toxicity.

An ideal strategy for postoperative management of CRC should simultaneously achieve three fundamental goals: precision targeting, therapeutic efficacy, and safety for quality of life.

Figure 8: Key design principles of RectomeDy FotoZymogen, emphasizing precision targeting, therapeutic efficacy, and safety with quality of life.

Thus, a truly innovative postoperative therapy for CRC should integrate localization, temporal controllability, molecular specificity, and redundant biosafety. Such a solution would provide high-intensity yet low-toxicity treatment precisely where it is needed, overcoming the limitations of systemic chemotherapy and radiotherapy. By ensuring both clinical efficacy and patient comfort, this new paradigm of postoperative intervention could redefine the balance between therapeutic power and life quality, paving the way toward a safer, smarter, and more sustainable future in colorectal cancer care.

The RDFZ team is dedicated to applying the power of synthetic biology to postoperative adjuvant therapy for colorectal cancer. We designed RectomeDy FotoZymogen, a “red-light–induced, site-specific bacterial therapy system” based on the probiotic strain E. coli Nissle 1917 (EcN). This system achieves full-process intelligent control — from precise localization, to efficient therapy, and finally to biological safety.

Figure 9: Workflow schematic of the RectomeDy FotoZymogen therapeutic cycle, from red-light activation to bacterial self-clearance.

We selected Escherichia coli Nissle 1917 (EcN) as the chassis organism for our project due to its well-established advantages in both synthetic biology and clinical applications. EcN is a probiotic strain with over a century of documented clinical safety in Europe, demonstrating excellent biocompatibility. Moreover, EcN naturally exhibits tumor-tropic behavior, actively migrating toward and accumulating within the hypoxic regions of colorectal tumors, providing a strong foundation for localized delivery and targeting. In addition, EcN possesses a mature and efficient genetic toolbox, allowing the convenient integration of complex synthetic biology circuits required for precise regulation and therapeutic control.

Figure 10: Scanning electron microscopic images, showing Escherichia coli strain Nissle 1917.showing retention of flagellae (f) and fimbriae (white arrowheads). Scale bars: 2 µm.[13]

Our RectomeDy FotoZymogen is a highly modular engineered bacterial system, integrating four core functional modules to achieve an intelligent closed-loop of biotherapeutic control. The engineered bacteria first utilize the targeted colonization module to achieve spatially precise localization at the tumor site. Under the control of the external red-light regulation system, the dual-payload synergistic therapy module is then activated to express therapeutic molecules on demand. Finally, the biosafety self-destruction system ensures that the engineered bacteria self-eliminate once their therapeutic mission is complete. Together, these four coordinated modules form a safe, efficient, and precise adjuvant treatment strategy for postoperative colorectal cancer therapy.

Figure 11: Schematic diagram of RectomeDy FotoZymogen’s modular system, integrating targeted colonization, red-light control, dual-payload therapy, and biosafety self-destruction.

Traditional adjuvant chemotherapy is systemic in nature, distributing cytotoxic agents throughout the entire body with limited tumor specificity. In contrast, our engineered probiotic functions like a biological precision-guided missile, navigating directly toward colorectal tumor residues through a molecularly defined targeting system.

We achieved tumor-specific adhesion using a bacterial surface display strategy, which enables functional proteins to be anchored on the outer membrane of E. coli Nissle 1917 (EcN). Specifically, we employed a truncated fragment of the Ice Nucleation Protein (INP) as an anchoring domain. INP is a well-characterized outer membrane protein capable of presenting heterologous peptides extracellularly without disrupting bacterial viability or surface integrity [13]. By fusing INP with a tumor-recognizing adhesin, we ensured that our targeting motif is stably and efficiently displayed on the bacterial surface, maintaining functionality within the intestinal microenvironment.

As the core adhesion ligand, we utilized Hemolysin-like Protein A (HlpA), a lectin-like protein known for its ability to specifically recognize Heparan Sulfate Proteoglycans (HSPGs), which are markedly overexpressed on colorectal cancer (CRC) cell membranes compared to normal epithelial cells. Literature reports indicate that the expression of HSPG in CRC tissues can be dozens of times higher than in adjacent healthy mucosa [14][15], providing an ideal molecular handle for selective colonization.

By combining INP-mediated surface anchoring with HlpA-based molecular recognition, our engineered EcN achieves high-affinity binding to CRC cells within the tumor microenvironment (TME). This enables the strain to selectively colonize regions of residual tumor tissue while minimizing interaction with normal intestinal surfaces. For consistent targeting performance during the colonization phase, the medium-strength constitutive promoter PJ23100 was chosen to drive steady expression of the INP–HlpA fusion protein, ensuring robust surface display without imposing excessive metabolic burden on the bacterial host.

This INP–HlpA adhesion system establishes the foundation for localized, precise, and controllable therapeutic intervention, enabling downstream modules to operate exclusively within the disease site and representing the first step toward safe and programmable post-surgical CRC therapy.

Figure 12: Schematic illustration of the adhesion system. After suppository dissolution, engineered probiotic E. coli Nissle 1917 expressing the INP–HlpA fusion protein specifically adheres to colorectal cancer cells via HSPG recognition on the tumor surface. The diagram depicts the localized targeting process within the colon and the molecular interaction between HlpA (on EcN surface) and HSPG (on tumor cell membrane), establishing a stable colonization at the tumor site for subsequent therapeutic activation.

Achieving true therapeutic precision requires not only spatial targeting but also temporal control. Conventional induction systems in synthetic biology, such as IPTG or arabinose-based triggers, suffer from poor tissue permeability, rapid metabolic clearance, and a lack of non-invasive controllability. To overcome these limitations, we integrated optogenetic regulation into our design, ensuring that the engineered bacteria activate their therapeutic functions only under externally applied red-light stimulation.

We adopted the Nano Engineered Temporal Modulation Activation Platform (NETMAP),[16], an advanced red/near-infrared (NIR) light–responsive system originally reported in Nature Cancer on 14 February 2025 (“Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics”). This platform provides a robust and reversible means of linking external illumination to gene expression in living bacteria.

In our construct, 660 nm red light is absorbed by the photoreceptor PadC, which undergoes a light-induced conformational transition that catalyzes the conversion of GTP into the secondary messenger c-di-GMP. This process effectively translates optical input into a biochemical signal. The intracellular accumulation of c-di-GMP subsequently activates the effector protein MrkH, which binds to its cognate promoter PmrkA, driving the expression of downstream therapeutic modules. These include Coa, a coagulase that promotes localized clot formation and microvascular blockade, and the PD-L1 nanobody (PD-L1 nb), which relieves immune suppression within the tumor microenvironment.

Figure 13: 660 nm red light is absorbed by NETMAP photoreceptors, converting the optical input into biochemical signals. This drives the expression of downstream therapeutic modules Coa and PD-L1nb.

To maintain minimal background activity in the absence of light, we co-expressed YhjH, a c-di-GMP phosphodiesterase, which continuously hydrolyzes basal c-di-GMP, ensuring that the circuit remains tightly repressed in the dark state. The BphO enzyme, co-expressed under the same regulatory network, synthesizes biliverdin (BV), the required chromophore for PadC activation, enabling autonomous operation within E. coli Nissle 1917.

According to the Nature Cancer (2025) study, the NETMAP system demonstrated a 55.1-fold increase in transcriptional activation under NIR illumination, with rapid response kinetics (significant activation within 30 minutes) and a tunable dose–time profile. These results underscore the superior precision and responsiveness of this optogenetic system compared with traditional chemical inducers. We adopted the same design principles to achieve fast, reversible, and quantitative control of therapeutic expression in vivo.

Furthermore, the use of 660 nm light—falling within the biologically safe NIR-I window—offers enhanced tissue penetration and minimal phototoxicity, overcoming the attenuation limits associated with blue or green light. This wavelength range ensures that illumination can reach the engineered bacteria residing within the colorectal lumen, even through layers of intestinal tissue and mucus.

To ensure the system’s clinical practicality, we integrated a miniaturized red-light LED module directly into the rectal suppository device, powered by a low-voltage circuit and controlled through a magnetic Hall switch. This configuration allows non-invasive wireless activation and immediate light delivery to the target region.

Figure 14: This is an introduction to our suppository. Left: The initial prototype, slightly thicker in size, featured two structural variants: one with stearic acid at the head and another with stearic acid at the tail.Head-type designs offered smoother and more comfortable insertion due to the soft stearic acid, but the epoxy edges at both ends created minor roughness.Tail-type designs had a smoother resin front, though they required glycerin lubrication during insertion since epoxy cannot self-lubricate like stearic acid.Both versions used two LED modules.The refined model on the right is slimmer, with a diameter of about 1 cm, making it much better suited for suppository delivery. For a more detailed version, please refer to our Hardware section.

In addition, we coupled the NETMAP system with a lyophilized (freeze-dried) bacterial formulation to achieve dual-layered control over therapeutic activation. While the red-light system remains active upon implantation, the engineered bacteria stay metabolically dormant in their lyophilized state, preventing premature expression. Only after rehydration by intestinal fluids and restoration of bacterial metabolism does the NETMAP circuit become fully responsive to red light, ensuring precise on-demand initiation of therapeutic protein expression.

This synergistic combination of optogenetic precision, hardware integration, and metabolic gating provides an unprecedented level of control over therapeutic gene activation, laying the foundation for a spatiotemporally confined, programmable, and clinically viable probiotic therapy for postoperative colorectal cancer management.

Figure 15: Schematic of the NETMAP-based red-light control system. The suppository releases engineered E. coli Nissle 1917 containing the NETMAP optogenetic circuit, which translates 660 nm red-light signals into c-di-GMP–mediated transcriptional activation. Upon illumination, PadC converts GTP to c-di-GMP, which activates MrkH binding to PmrkA, initiating expression of therapeutic effectors Coa and PD-L1 nanobody. The system’s design was adapted from Nature Cancer (14 February 2025), “Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics”.

The core of our therapeutic module lies in a dual-pronged, synergistic killing strategy, where two mechanistically distinct effectors are co-expressed and released exclusively under NETMAP activation in the localized tumor microenvironment. This ensures that both components achieve spatial precision, temporal synchronization, and functional complementarity to maximize antitumor efficacy while preserving biosafety.

Payload 1: Coagulase (Coa)

We selected coagulase (Coa) as the first therapeutic payload due to its unique ability to induce localized vascular coagulation within the tumor microenvironment (TME). Coa is a secreted enzyme that activates the host’s prothrombin-to-thrombin cascade, converting fibrinogen into fibrin and thereby generating a dense coagulative barrier surrounding the tumor tissue. This fibrin network effectively blocks microvascular blood flow, depriving tumor cells of nutrients and oxygen, ultimately leading to tumor starvation and necrosis [17][18].

Beyond direct cytotoxicity, this coagulative encapsulation also serves as a physical biosafety barrier, preventing engineered bacteria or therapeutic molecules from entering systemic circulation. By spatially confining the therapeutic agents, Coa reduces the risk of systemic leakage and minimizes potential immunotoxicity. Previous research has demonstrated that localized coagulation can enhance both tumor suppression and safety, making Coa an ideal component of our biosafe therapeutic design（Zou et al., Advanced Science, 2025）.

Payload 2: Anti–PD-L1 Nanobody (PD-L1 nb)

Colorectal cancer cells are known to overexpress the immune checkpoint ligand PD-L1, which binds to PD-1 receptors on activated T cells, transmitting an inhibitory signal that suppresses immune activity — a mechanism often referred to as an “immune brake.” Our anti–PD-L1 nanobody is designed for high local expression and efficient neutralization of PD-L1, thereby releasing this immune brake and restoring cytotoxic T-cell function. Unlike systemic monoclonal antibody therapies, the nanobody format provides smaller molecular size, higher tissue penetration, and lower immunogenicity, making it particularly suitable for localized, probiotic-based delivery [19][20].

Synergistic Mechanism of Action: The combination of Coa-induced coagulation and PD-L1 blockade produces a complementary “kill-and-alert” therapeutic cascade. Coa-mediated vascular occlusion leads to tumor cell necrosis, causing the release of tumor-associated antigens (TAAs) into the surrounding microenvironment. Simultaneously, the PD-L1 nanobody ensures that effector T cells remain active and unrestrained, enabling them to recognize and attack the newly exposed tumor antigens. This interlocked synergy not only enhances immediate tumor clearance but also promotes the establishment of long-term immune memory, potentially reducing postoperative recurrence of CRC.

Figure 16: Schematic illustration of the dual-therapeutic payload system.Left: Upon red-light activation, engineered E. coli Nissle 1917 expresses and releases two therapeutic agents—coagulase (Coa) and anti–PD-L1 nanobody (PD-L1 nb)—within the colorectal tumor microenvironment.Upper Right: Diagram showing the mechanism of Coa, which converts prothrombin into thrombin to form fibrin clots, creating a localized coagulative barrier that cuts off tumor blood supply and prevents systemic leakage of engineered bacteria.Lower Right: Mechanism of PD-L1 nanobody action, where blockade of PD-L1 restores T-cell cytotoxic function, enabling immune-mediated tumor cell killing and promoting immune memory formation.

Together, this dual-payload architecture achieves multi-layered precision therapy—spatially confined by targeted colonization, temporally gated by red-light induction, and biologically amplified through immune reactivation—representing a safe, powerful, and clinically adaptable model for adjuvant colorectal cancer treatment.

We consider biosafety the non-negotiable precondition for any living therapeutic. To provide high-redundancy containment while preserving operational flexibility during treatment, we designed the ORION Kill-Switch, a three-input, OR-logic, toxin/antitoxin–based safety module that enforces programmed death of the therapeutic chassis whenever both external survival signals are absent. The architecture deliberately separates a constitutive toxin baseline from signal-dependent antitoxin control, such that continued bacterial viability requires at least one exogenous survival input (red-light or L-arabinose). This arrangement yields clear, clinically useful control: during therapy the strain is maintained alive by light and/or arabinose; after therapy both signals can be removed to trigger rapid, autonomous elimination [21][22].

Figure 17: Schematic illustration of the ORION kill-switch mechanism, showing bacterial survival under red light or arabinose induction and programmed self-destruction when both signals are absent.

Design overview

• Toxin effector:mazF (mRNA endonuclease) is driven by a promoter with measurable basal leak (e.g., P___SUB___lac___). Low-level, constitutive MazF expression establishes a persistent toxic baseline that would kill the cell unless neutralized by antitoxin.
• Antitoxin controllers:mazE expression is placed under two independent, orthogonal inducible promoters: P___SUB___BAD___ (L-arabinose inducible) and P___SUB___mrkA___ (NETMAP red-light inducible via MrkH). mazE can therefore be produced in response to either L-arabinose or red light.
• Logical behavior: The system implements an OR gate for survival: presence of (arabinose OR red light) → mazE ON → MazF neutralized → cell survives; absence of both → mazE OFF → MazF activity dominates → programmed death.
• Operational controls: During manufacturing and therapeutic application, external provision of L-arabinose (in the patient’s oral intake or suppository formulation) and/or local red-light illumination maintains mazE expression. After device removal and cessation of arabinose supply, the antitoxin reservoir decays rapidly and MazF toxicity is unleashed, producing robust bacterial self-clearance in the hours following signal withdrawal.

Why this topology?

• High redundancy: Two independent antitoxin inputs (chemical and optical) reduce single-point failure risk and give clinicians flexible control modes (hardware-only, diet-supplemented, or both).
• Fail-safe elimination: Constitutive low-level toxin ensures that loss of all exogenous inputs leads predictably to death rather than to ambiguous dormancy.
• Clinical practicality: P___SUB___BAD___ (arabinose) is safe, non-toxic, and can be provided orally; red light is non-invasive and locally confined by the suppository device. Combining both allows in-clinic or at-home maintenance, and an easy switch-off by stopping either input (particularly stopping arabinose ingestion and removing illumination).
• Regulatory friendliness: The architecture provides an auditable, temporally bounded survival window and a demonstrable, quantitative kill profile — features that facilitate biosafety review.

2×2 Decision Table — ORION Kill-Switch Outcomes

Table 2: ORION Kill-Switch Outcomes

The RectomeDy FotoZymogen system integrates all functional modules into a precisely regulated, modular therapeutic framework, achieving spatial targeting, temporal control, and built-in biosafety through a closed-loop design.

In the encapsulation stage, engineered E. coli Nissle 1917 is lyophilized into dormancy, with L-arabinose–induced MazE expression counteracting basal MazF toxicity to ensure high viability and long-term stability.

During colonization, rehydrated bacteria express INP–HlpA, enabling specific adhesion to colorectal tumor cells via HSPG recognition and achieving accurate spatial localization in the tumor microenvironment.

In the activation stage, the 660 nm red-light NETMAP circuit converts optical input into c-di-GMP signaling, activating PmrkA to drive high-level expression of Coa, PD-L1 nanobody, and MazE, ensuring precise, on-demand therapeutic induction.

The synergistic therapy phase follows: Coa forms a localized fibrin barrier to block tumor blood supply, while the PD-L1 nanobody restores T-cell activity, achieving “vascular blockade + immune activation” for efficient tumor clearance.

Finally, in the self-clearance stage, the absence of red light and L-arabinose triggers MazE degradation and MazF-mediated bacterial self-destruction, ensuring automatic elimination post-treatment.

Figure 18: Conceptual workflow of RectomeDy FotoZymogen within the colorectal tract, demonstrating targeting, activation, tumor killing, and final bacterial clearance.

Overall, RectomeDy FotoZymogen represents a programmable, self-contained living medicine, offering safe, localized, and controllable postoperative therapy for colorectal cancer recurrence prevention.

1. Target Customers

Our target population consists of patients who have undergone surgical resection for colorectal cancer (CRC) and remain at high risk of postoperative recurrence or metastasis. These individuals typically present with minimal residual disease (MRD) that cannot be fully removed through surgery and thus require safe, localized adjuvant therapy to prevent recurrence. The system is specifically designed for postoperative maintenance treatment, providing a controllable and patient-friendly solution that complements existing chemotherapy or radiotherapy regimens while minimizing systemic side effects.

2. Production and Development

In real-world implementation, the biological component will be mass-produced as a lyophilized probiotic powder, ensuring long-term stability and storage convenience. The engineered E. coli Nissle 1917 will be cultured under controlled bioreactor conditions and freeze-dried into sealed capsules that maintain viability until use. In parallel, the hardware component will be manufactured as a single-use rectal suppository device integrating a 660 nm LED module, microcontroller, and magnetic activation switch. This disposable design simplifies hygiene management, eliminates reuse risks, and supports straightforward clinical distribution. The complete product — a biologically active suppository with integrated illumination — can be distributed through hospitals, oncology centers, or prescription-based retail channels, enabling physician-guided application in postoperative care.

Figure 19: Simplified overview of production and development, from probiotic powder preparation to single-use suppository manufacturing and clinical distribution.

3. Method of Use

Patients will receive the system in a sterile package. To use, they simply remove the protective film, attach the small magnetic activator to power on the internal red-light source, and gently insert the suppository into the rectum. Once in place, the suppository dissolves upon contact with intestinal fluid, rehydrating the lyophilized engineered bacteria and initiating light-induced therapeutic activation. During illumination, the bacteria express Coa and PD-L1 nanobody, performing localized coagulation and immune reactivation. After several hours, the suppository and bacteria are naturally expelled during defecation, concluding one treatment cycle. The procedure is non-invasive, self-administered, and compatible with standard postoperative routines, allowing repeated or physician-scheduled applications as needed.

Figure 20: Step-by-step usage instructions of the red-light suppository system, including activation, insertion, and natural expulsion after treatment.

4. Safety Considerations

Patient and environmental safety remain central to our implementation plan. The engineered strain is based on E. coli Nissle 1917, a clinically recognized probiotic. The system incorporates dual-layer biosafety protection: (1) the NETMAP optogenetic circuit, which confines therapeutic activation strictly to red-light exposure, and (2) the ORION kill-switch, ensuring bacteria undergo programmed self-elimination once both arabinose and light signals are absent. This guarantees that, after excretion, the bacteria cannot survive in the external environment. Furthermore, the hardware is fully biocompatible and disposable, minimizing cross-contamination risk. Collectively, these safety measures provide predictable containment, controlled activity, and rapid clearance, ensuring compliance with future clinical and regulatory biosafety standards.

Figure 21: Summary of the six major advantages of RectomeDy FotoZymogen, emphasizing precision, controllability, safety, and scalability.

In the next stage of development, we will focus on advancing RectomeDy FotoZymogen toward clinical translation and real-world application. Our immediate goals include optimizing optogenetic response sensitivity and payload release dynamics, conducting comprehensive preclinical evaluations to verify biosafety and therapeutic efficacy in animal models, and further enhancing device miniaturization to improve patient comfort and usability.

Looking ahead, we envision RectomeDy FotoZymogen as more than a single therapeutic product—it represents a transformative paradigm for precision biomedicine. By integrating programmable biology with non-invasive hardware, this platform has the potential to pioneer a new generation of living medicines capable of intelligent sensing, targeted intervention, and self-regulation. Beyond colorectal cancer, we aspire to expand its application to other gastrointestinal malignancies and localized chronic diseases, ultimately contributing to a future where biological therapy is precise, personalized, and accessible to all.

1. O’Connor, E. S., Greenblatt, D. Y., LoConte, N. K., et al. (2011). Adjuvant chemotherapy for stage II colon cancer with poor prognostic features. Journal of Clinical Oncology, 29(25), 3381–3388. https://doi.org/10.1200/JCO.2010.33.1792
2. Andre, T., Boni, C., Mounedji-Boudiaf, L., et al. (2004). Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. New England Journal of Medicine, 350(23), 2343–2351. https://doi.org/10.1056/NEJMoa032709
3. Van Cutsem, E., Cervantes, A., Adam, R., et al. (2016). ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Annals of Oncology, 27(8), 1386–1422. https://doi.org/10.1093/annonc/mdw235
4. Senevirathne, A., Lloren, K. K. S., Aganja, R. P., et al. (2025). Transforming bacterial pathogens into wonder tools in cancer immunotherapy. Molecular Therapy, 33(2). https://doi.org/10.1016/j.ymthe.2025.01.003
5. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide. CA: A Cancer Journal for Clinicians, 68(6), 394–424. https://doi.org/10.3322/caac.21492
6. Dekker, E., Tanis, P. J., Vleugels, J. L. A., Kasi, P. M., & Wallace, M. B. (2019). Colorectal cancer. The Lancet, 394(10207), 1467–1480. https://doi.org/10.1016/S0140-6736(19)32319-0
7. Van Cutsem, E., Cervantes, A., Adam, R., et al. (2016). ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Annals of Oncology, 27(8), 1386–1422. https://doi.org/10.1093/annonc/mdw235
8. Tarazona, N., Gimeno-Valiente, F., Gambardella, V., et al. (2021). Circulating tumor DNA in colorectal cancer. Advances in Clinical Chemistry, 102, 71–119. https://doi.org/10.1016/bs.acc.2020.10.004
9. O'Connor, E. S., Greenblatt, D. Y., LoConte, N. K., et al. (2011). Adjuvant chemotherapy for stage II colon cancer with poor prognostic features. Journal of Clinical Oncology, 29(25), 3381–3388. https://doi.org/10.1200/JCO.2010.33.1792
10. Andre, T., Boni, C., Mounedji-Boudiaf, L., et al. (2004). Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. New England Journal of Medicine, 350(23), 2343–2351. https://doi.org/10.1056/NEJMoa032709
11. Franco, P., Arcadipane, F., Ragona, R., et al. (2020). Intestinal toxicity after pelvic radiotherapy: A multidisciplinary approach. World Journal of Gastroenterology, 26(16), 1684–1698. https://doi.org/10.3748/wjg.v26.i16.1684
12. Fakih, M. (2015). Metastatic colorectal cancer: Current state and future directions. Journal of Clinical Oncology, 33(16), 1809–1824. https://doi.org/10.1200/JCO.2014.60.9217
13. Barisani-Asenbauer, T., Montanaro, J., Inic-Kanada, A., Ladurner, A., Stein, E., Belij, S., Bintner, N., Schlacher, S., Schuerer, N., Mayr, U. B., Lubitz, W., & Leisch, N. (2015). Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: An imaging study of a delivery system for the ocular surface. Drug Design, Development and Therapy, 3741. https://doi.org/10.2147/DDDT.S84370
14. Ho, C. L., Tan, H. Q., Chua, K. J., Kang, A., Lim, K. H., Ling, K. L., & Yew, W. S. (2018). Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature Biomedical Engineering, 2(1), 27–37. https://doi.org/10.1038/s41551-017-0181-y
15. Ma, X., Liang, X., Li, Y., Feng, Q., Cheng, K., Ma, N., … & Shi, J. (2023). Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field. Nature Communications, 14, Article 1471. https://doi.org/10.1038/s41467-023-37225-1
16. Kwon, S. Y., Ngo, H. T.-T., Son, J., Hong, Y., Kim, S. B., & Park, Y. (2024). Exploiting bacteria for cancer immunotherapy. Nature Reviews Clinical Oncology, 21, 198–213. https://doi.org/10.1038/s41571-024-00908-9
17. Qiao, L., Niu, L., Wang, Z. et al. Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics. Nat Cancer 6, 612–628 (2025). https://doi.org/10.1038/s43018-025-00932-3
18. Wang, W., Liu, L., You, L., et al. (2021). Tumor microenvironment-activated coagulase nanoparticles for cascade targeting and synergistic tumor therapy. ACS Nano, 15(9), 14370–14382. https://doi.org/10.1021/acsnano.1c02703
19. Zhang, L., Li, Y., Yu, J., et al. (2020). Locally injectable hydrogel–based coagulase system for tumor-specific vascular disruption. Science Translational Medicine, 12(531), eaax9380. https://doi.org/10.1126/scitranslmed.aax9380
20. Hassanzadeh, A., Hossini, A. M., & Ebrahimi, M. (2023). Nanobody-based targeting of PD-L1 in colorectal cancer: Advances and challenges. Frontiers in Oncology, 13, 1081570. https://doi.org/10.3389/fonc.2023.1081570
21. Bannas, P., Hambach, J., & Koch-Nolte, F. (2017). Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Frontiers in Immunology, 8, 1603. https://doi.org/10.3389/fimmu.2017.01603
22. Stirling, F., Bitzan, L., O’Keefe, S., et al. (2017). Rational design of evolutionarily stable microbial kill switches. Molecular Cell, 68(4), 686–697.e3. https://doi.org/10.1016/j.molcel.2017.10.033
23. Chan, C. T. Y., Lee, J. W., Cameron, D. E., et al. (2016). ‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment. Nature Chemical Biology, 12, 82–86. https://doi.org/10.1038/nchembio.1979