A tumor diagnosis and treatment kit, a preparation method and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and specifically relates to a tumor diagnosis and treatment kit, a preparation method and application thereof. The present application creatively uses genetically engineered bacteria expressing lactoferrin and enteric iron source together in the diagnosis and treatment of tumors. Iron ions are recruited and fixed in situ by lactoferrin in the tumor microenvironment, thereby constructing a sustained tumor iron depot in the tumor local area, which can synergistically induce tumor cell ferroptosis and activate systemic anti-tumor immune response, has the effects of high efficiency and low toxicity, and has good popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a tumor diagnostic and treatment reagent kit, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, with advanced recurrence, distant metastasis, and drug resistance severely impacting long-term survival. While significant progress has been made in immune checkpoint blockade (ICB) therapy in recent years, response rates to immunotherapy remain low for approximately 85% of microsatellite stable (MSS) CRC cases due to insufficient immune cell infiltration in tumor tissue, a significant immunosuppressive microenvironment, and low antigen presentation efficiency. Therefore, there is an urgent need to develop novel therapeutic strategies capable of reshaping the tumor immune microenvironment.

[0003] Ferroptosis is a novel programmed cell death mechanism that relies on iron ions to catalyze lipid peroxidation. It not only directly kills tumor cells but also induces immunogenic cell death (ICD), promoting the release of damage-related molecules such as high-mobility group box 1 (HMGB1), adenosine triphosphate (ATP), and calreticulin (CRT), further promoting dendritic cell maturation and... Cell activation is therefore considered an important pathway to enhance the efficacy of tumor immunotherapy. However, ferroptosis is highly dependent on a continuous and sufficient supply of iron to the tumor site, while iron metabolism in the human body is strictly regulated, resulting in extremely low levels of free iron. This means that current ferroptosis therapies typically require exogenous iron supplementation or delivery via iron nanomaterials. Currently, iron nanoparticles, iron ion complexes, and various siderophores are used to enhance ferroptosis; for example, Ren et al. used orlistat with… A self-assembled metal-coordinated nanoparticle, OFe-NPs, exhibited GSH-responsive dissociation properties in MCF-7 breast cancer cells, achieving a tumor inhibition rate of 76.76% in a tumor-bearing mouse model (Cancer Nanotechnology, 2026, 17, 26). The axially chlorine-coordinated iron single-atom nanozyme, FeN4Cl, designed by Yin et al., showed a 2.0-fold increase in peroxidase-like activity and a 3.2-fold increase in glutathione oxidase-like activity. It also established an autonomous iron supply mechanism by triggering NCOA4-mediated ferritin autophagy, and its biocompatibility and tumor targeting were enhanced after encapsulation in erythrocyte membranes (Journal of Nanobiotechnology, 2026, 24, 301). Regarding iron ion complexes, Chen et al. reported that the hino-iron complex Fe(hino)3, at moderate doses, induces ferroptosis in osteosarcoma by disrupting mitochondrial function, enhancing ROS generation, and lipid peroxidation; at high doses, it activates the NLRC4 / Caspase-1 / GSDMD pathway to induce pyroptosis (International Journal of Biological Sciences, 2025, 21, 11, 4872–4894). Iron-based MOFs, such as bortezomib@RhB-MOF (BTZ@RhB-MOF) developed by Lu et al., target the mitochondrial outer membrane protein TOM20 in colorectal cancer, inducing ferroptosis by releasing iron ions (Molecular Pharmaceutics, 2025, 22, 8, 4865–4878). Q-MIL-53(Fe) nanozymes activate anti-tumor immune responses by inducing ferroptosis and immunogenic cell death, promoting dendritic cell maturation and T lymphocyte infiltration (Nature Communications, 2025). 16,2290). Furthermore, ferritin carriers, such as the ferritin nanocages co-encapsulated with iron and siGPX4 developed by Professor Cui Chunying's team (HFn@Fe / siGPX4), utilize the active targeting of ferritin to precisely deliver drugs to the tumor region. Iron ions trigger the Fenton reaction, inducing ferroptosis, and neutralize the negative charge of siGPX4, promoting its loading and lysosomal escape (ActaPharmaceutica Sinica B, 2024). The transferrin-modified liposomes loaded with gambogeylic acid developed by Rong et al. (GA@Tf-Lip) leverage the high expression of transferrin receptors in tumor cells to simultaneously achieve tumor targeting and Fe³⁺ carrier function, increasing intracellular iron ion content and promoting the Fenton reaction (Scientific Reports, 2026). However, these strategies generally suffer from insufficient tumor targeting, short circulation time, difficulty in maintaining local iron concentration, and high systemic toxicity.Furthermore, most siderophores can only release iron once and cannot sustainably maintain the iron-rich state in the tumor, thus limiting the therapeutic effect of ferroptosis, even if. These systems that attempt to self-supply iron still face challenges in terms of in vivo delivery efficiency and long-term safety. How to sustainably and efficiently supply iron for cancer treatment is a topic worthy of further research in this field. Summary of the Invention

[0004] To address the goal of continuously and efficiently delivering iron to tumor tissues in vivo to activate tumors and enhance the efficacy of tumor immunotherapy, this invention, through long-term research, has discovered that by utilizing engineered probiotics to continuously express lactoferrin in situ, a stable iron recruitment system can be established at the tumor site. Combined with iron-based preparations to continuously supplement iron ions, a sustained local iron reservoir can be formed, achieving long-term induction of ferroptosis and enhanced anti-tumor immunity. This completes the present invention, and the technical solution is as follows:

[0005] In a first aspect, the present invention discloses a tumor diagnostic and therapeutic kit, the kit comprising engineered bacteria and an enteric iron source preparation; the engineered bacteria are genetically engineered to secrete and express lactoferrin.

[0006] In this invention, lactoferrin recruits and immobilizes iron ions in situ in the tumor microenvironment, thereby constructing a persistent tumor iron reservoir in the tumor site to synergistically induce ferroptosis in tumor cells and activate a systemic anti-tumor immune response.

[0007] In some embodiments, the chassis bacteria of the engineered bacteria are selected from strains with tumor-targeting ability, intestinal colonization ability, or barrier-penetrating ability.

[0008] In one embodiment, the bacteria in the substrate are selected from attenuated Salmonella, Bacillus subtilis, Escherichia coli, lactic acid bacteria, Bifidobacterium, Clostridium, and Listeria. Preferably, the bacteria in the substrate are selected from Escherichia coli Nissle 1917 (EcN) or attenuated Salmonella VNP20009.

[0009] In some embodiments, the engineered bacteria co-express not only lactoferrin but also adjunctive therapeutic proteins.

[0010] In some embodiments, the adjuvant therapeutic protein is selected from one or more of cytokines, immunomodulatory proteins, chemokines, antibodies, nanobodies, or enzymes.

[0011] In one embodiment, the adjuvant therapeutic protein is selected from one or more of interferon-γ (IFN-γ), interleukin-12 (IL-12), interleukin-15 (IL-15), PD-L1 nanobody, CTLA-4 nanobody, and glucose oxidase.

[0012] In some embodiments, the enteric iron source formulation is selected from iron salts or iron chelates coated with an enteric coating.

[0013] In one embodiment, the iron salt or iron chelate is selected from one or more of ferrous gluconate, ferrous succinate, ferrous fumarate, sodium iron EDTA, iron dextran, and ferric ammonium citrate.

[0014] The diagnostic and treatment methods described in this invention include diagnosis and / or treatment, meaning that the diagnostic and treatment kits of this invention can be used for both the diagnosis and treatment of tumors.

[0015] In this invention, the diagnosis is made by collecting symptoms, signs and examination results, and analyzing and judging the nature and state of the disease.

[0016] In this invention, the treatment is to eliminate or reduce disease or injury and improve bodily function through intervention.

[0017] In this invention, the enteric iron source formulation with enteric coating does not release iron in the acidic environment of the stomach, but dissolves and releases iron ions in the alkaline environment of the intestine, so as to efficiently recruit lactoferrin secreted by engineered bacteria that colonize the tumor site.

[0018] In this invention, lactoferrin (Lf), as a natural iron-binding glycoprotein, possesses extremely high iron-binding capacity and good biocompatibility, and can bind with... It forms a stable complex and maintains high iron-binding activity in a weakly acidic environment. Because tumor tissue is in a chronically acidic microenvironment, lactoferrin can continuously recruit circulating iron sources, creating an iron-rich environment in the tumor. Simultaneously, lactoferrin can regulate the function of dendritic cells, macrophages, and NK cells through signaling pathways such as TLR4 and lactoferrin receptor (LfR), acting as a natural immune adjuvant. In this invention, the engineered bacteria express lactoferrin, enabling them to recruit and fix iron ions in situ within the tumor microenvironment, thereby constructing a persistent tumor iron reservoir in the tumor site. This synergistically induces ferroptosis in tumor cells and activates a systemic anti-tumor immune response. Specifically, the iron ions enriched in the tumor iron reservoir react with endogenous hydrogen peroxide within tumor cells via a Fenton reaction, producing highly toxic reactive oxygen species (ROS). This induces ferroptosis in tumor cells and releases tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), thereby activating dendritic cell maturation, promoting T cell infiltration, transforming "cold" tumors into "hot" tumors, and activating anti-tumor therapy.

[0019] In this invention, the highly enriched iron in the persistent tumor iron reservoir constructed locally in the tumor produces a significant magnetic resonance (MRI) contrast enhancement effect in the tumor. Therefore, the kit of this invention can be used for real-time, non-invasive monitoring of the spatial distribution of the engineered bacteria colonization in the tumor site, the iron recruitment kinetics, and the response status of tumor treatment.

[0020] In a second aspect, the present invention discloses a method for preparing a tumor diagnostic and therapeutic reagent kit, the method comprising:

[0021] (1) Construct a recombinant plasmid containing a lactoferrin expression cassette and transform it into a chassis host bacterium to screen for recombinant engineered bacteria that can stably secrete and express lactoferrin;

[0022] (2) The recombinant engineered bacteria are combined with the enteric iron source preparation to obtain the product.

[0023] In one embodiment, the method further includes coating the iron source with an enteric material to obtain an enteric iron source formulation.

[0024] In one embodiment, the recombinant engineered bacteria also co-express the aforementioned adjuvant therapeutic protein of the present invention.

[0025] In a third aspect, the present invention discloses the use of the tumor diagnostic kit of the first aspect of the present invention in the preparation of medicaments for the prevention, treatment or diagnosis of tumors.

[0026] In some embodiments, the tumor is a solid tumor.

[0027] In one embodiment, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, and liver cancer; preferably, the tumor is selected from colorectal cancer, and more preferably, the colorectal cancer is microsatellite stable (MSS) colorectal cancer.

[0028] In a fourth aspect, the present invention discloses a method for diagnosing and treating tumors, the method comprising administering a tumor diagnosis and treatment kit of one aspect of the present invention to a subject.

[0029] In this invention, the tumor diagnostic and therapeutic kit is administered orally or intravenously; then, the engineered bacteria actively target and colonize the tumor lesion, continuously secreting and expressing lactoferrin; wherein, the enteric-coated iron source preparation is administered orally. After oral administration of the enteric-coated iron source preparation, iron ions are released at the intestinal lesion site, specifically recruited and fixed by the lactoferrin, establishing an in-situ tumor iron reservoir, achieving continuous ferroptosis, immune activation, and magnetic resonance imaging monitoring.

[0030] Beneficial effects of the present invention

[0031] (1) This invention breaks through the bottleneck of one-time iron supply and constructs a continuous "tumor iron reservoir" in situ: Traditional ferroptosis therapy relies on the type I release of nanoparticles or iron complexes, which cannot maintain long-term local iron accumulation. This invention utilizes the long-term colonization and activity of engineered bacteria in tumor lesions to continuously secrete lactoferrin with strong affinity in situ, constantly "capturing" and "fixing" exogenous enteric iron and endogenous circulating iron, constructing a dynamically balanced and continuously existing "iron reservoir" in the tumor site, and realizing long-term and controllable ferroptosis induction.

[0032] (2) This invention combines targeted delivery with enteric protection to reduce systemic toxicity: By combining the tumor active chemotaxis / targeting ability of engineered bacteria with the enteric coating protection of exogenous iron sources, the premature leakage of iron ions in the stomach and the risk of systemic iron overload are avoided, which greatly improves the local utilization efficiency of iron ions and reduces toxic side effects.

[0033] (3) This invention has the ability to efficiently induce ICD and reshape the "cold" tumor immune microenvironment: the tumor iron reservoir triggers a violent Fenton response, producing a large amount of ROS to promote ferroptosis, accompanied by CRT membrane flipping, HMGB1 release, and explosive ATP secretion (DAMPs). This significantly promotes the phenotypic maturation of dendritic cells (DCs) (CD80). + CD86 + and CD11c + MHC II + Upregulation) efficiently activates T cell responses, successfully transforming "cold" tumors such as microsatellite stable (MSS) colorectal cancer into immune-activated "hot" tumors.

[0034] (4) This invention eliminates the need for external contrast agents, enabling real-time monitoring of "integrated diagnosis and treatment": The highly enriched iron element in the tumor iron reservoir naturally possesses excellent magnetic resonance (MRI) contrast enhancement capabilities. Without the need for additional injection of highly toxic magnetic nanomaterials, it is possible to non-invasively, in real-time, and dynamically track the colonization and distribution of engineered bacteria in the body, the iron recruitment process, and the tumor treatment effect. Attached Figure Description

[0035] Figure 1 The structure map of the recombinant plasmid P21-RBS6-SPyycP-HLF-his expressing human lactoferrin constructed in this embodiment of the invention (the plasmid is 10,504 base pairs in length and is labeled with key elements such as promoter, signal peptide, HLF coding region, His tag and restriction cleavage site).

[0036] Figure 2The bar chart shows the ELISA quantitative detection results of human lactoferrin (HLF) secreted by recombinant Escherichia coli under different induction conditions / expression systems (VNP, T0~T5) in the embodiments of the present invention. , , , ns indicates no significant difference).

[0037] Figure 3 This is an SDS-PAGE protein gel electrophoresis identification image of the recombinant Escherichia coli expression product in the embodiments of the present invention (M is Protein Marker, lanes 1-8 are expression samples collected under different induction conditions, and the yellow dashed box indicates the target lactoferrin-specific band region).

[0038] Figure 4 This is a growth curve of blank Escherichia coli (E. coli) and recombinant expression plasmid engineered bacteria (E. coli-Plasmid) within 0-7 h in the embodiments of the present invention.

[0039] Figure 5 This is a flow cytometry statistical graph showing the phenotypic maturation and activation of dendritic cells (DCs) stimulated by recombinant engineered bacteria in an embodiment of the present invention; where a is... Proportional statistics chart, b represents antigen presentation function DC Proportional Statistical Chart ( , ns indicates no significant difference).

[0040] Figure 6 These are fluorescence micrographs showing the induced reactive oxygen species (ROS) production and oxidative stress in cells in the control group, the engineered bacteria treatment group alone, and the engineered bacteria combined with iron treatment group in the embodiments of the present invention.

[0041] Figure 7 This is a quantitative detection diagram of immunogenic cell death (ICD) and DAMPs release induced by engineered bacteria combined with iron treatment in an embodiment of the present invention; where a represents calreticulin on the cell membrane surface ( (Flip the scale diagram; b represents intracellular high-mobility group proteins) Residual ratio plot, c is the statistical plot of extracellular ATP release concentration ( , ).

[0042] Figure 8 These are in vivo magnetic resonance imaging (MRI) comparison images of tumor-bearing mice under different treatments (control group, blank E. coli + iron, and E. coli expressing lactoferrin) in this embodiment of the invention (top row is cross-sectional image, bottom row is coronal image).

[0043] Figure 9 This is a monitoring curve of weight change in mice in each group (control group, blank Escherichia coli + iron, and Escherichia coli expressing lactoferrin + iron) during a continuous 7-day treatment period in this embodiment of the invention.

[0044] Figure 10 This is a comparison of Kaplan-Meier survival curves and median survival time of tumor-bearing mice in each treatment group during long-term treatment in the embodiments of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0046] Example 1:

[0047] 1. Construction of recombinant plasmids, preparation of engineered bacteria, and validation of secretory expression.

[0048] 1) Construction and identification of recombinant plasmid P21-RBS6-SPyycP-HLF-his

[0049] Gene Synthesis and Cloning: Based on the amino acid sequence of human lactoferrin (HLF), codons for *E. coli* were optimized to synthesize the DNA sequence encoding the HLF protein, and a sequence encoding a 6×His tag was fused to its C-terminus. An expression vector backbone containing the ampicillin resistance gene (AmpR), replication origin (ori), P21 promoter, RBS6 ribosome binding site, and the SPyycP potent secretion signal peptide sequence was constructed. The HLF-his fragment was inserted into the vector via restriction endonuclease digestion and T4 DNA ligase ligation, resulting in the expression plasmid P21-RBS6-SPyycP-HLF-his (full length 10,504 bp, plasmid map shown). Figure 1 ).

[0050] Fusion Protein Optimization Design: To improve the expression efficiency, secretion capacity, and native biological activity of recombinant HLF, the HLF coding sequence was systematically optimized during gene design. While maintaining the amino acid sequence, codon optimization was performed based on the codon preferences of Bacillus subtilis, reducing GC content and mRNA secondary structure stability, minimizing consecutive rare codons and potential transcription termination sites, and improving translation efficiency. Simultaneously, a highly efficient secretion signal peptide, SPyycP, was fused to the N-terminus to guide the release of recombinant HLF into the extracellular space via the Sec secretion pathway; a 6×His tag was fused to the C-terminus for subsequent protein purification and detection by Western blot and ELISA. To avoid the tag's influence on the protein's native folding and function, a flexible linker peptide (such as GGGGS) was placed between the HLF and His tag, increasing the spatial freedom of the fusion protein and ensuring the structural integrity and iron-binding activity of lactoferrin.

[0051] Transformation and Recombinant Strain Screening: The validated recombinant plasmid was transformed into competent E. coli cells (E. coli Nissle 1917, EcN) using the heat shock method. The transformed plasmid was plated on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37 ℃ for 16 h. Single colonies were picked for somatic PCR verification and sequencing comparison to screen for the high-expression recombinant engineered strain E. coli-Plasmid.

[0052] 2) Quantitative analysis of lactoferrin (HLF) expression in engineered strains and optimization of induction conditions

[0053] Induction Culture and Supernatant Collection: The recombinant engineered strain was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm with shaking. It was then transferred to fresh LB medium at a 1:100 ratio and cultured until the OD600 reached 0.6–0.8. Different induction conditions / expression systems were then established (groups included: VNP control group, T0, T1, T2, T3, T4, and T5 groups, corresponding to different combinations of promoter strength, inducer concentration, and culture temperature). After culturing for another 12 h, the cells were centrifuged at 4°C and 10,000 xg for 15 min, and the clear cell supernatant was collected.

[0054] ELISA Quantitative Detection: The absolute secretion concentration of HLF in the supernatant was detected using a human lactoferrin (HLF) enzyme-linked immunosorbent assay (ELISA) kit. Figure 2As shown, in the VNP blank group and under T0 and T1 conditions, the detection value of HLF was extremely low or undetectable; with the optimization of the expression system and induction parameters (T2~T5 groups), the secretory expression level of HLF showed a significant increasing trend. Among them, under T5 induction conditions, the secretory expression level of HLF in the supernatant reached the highest level, approximately 1.5 x 10⁻⁶. 4 pg / mL (i.e. 15 ng / mL) indicates that the constructed system has a high efficiency in secreting exogenous proteins.

[0055] 3) Verification by SDS-PAGE protein gel electrophoresis

[0056] Sample preparation and electrophoresis: The culture supernatant from each of the above induction groups (lanes 1-8) was mixed with 5× SDS-PAGE loading buffer and heated at 95℃ for 10 min to denature. Electrophoresis was performed using a 10% polyacrylamide gel (constant voltage 120V, approximately 90 min), with Protein Molecular Weight Markers (M, molecular weight range 10-180 kDa) added to both sides. After electrophoresis, the samples were stained with Coomassie Brilliant Blue R-250 for 1 h and then destained with destaining solution until the background was clear.

[0057] Results Analysis: For example Figure 3 As shown, at a molecular weight of approximately 75 kDa (the area indicated by the yellow dashed box, consistent with the theoretical molecular weight of HLF), lanes 1-8 exhibit specific protein bands of corresponding intensities, and the band abundance is similar to... Figure 2 The ELISA quantitative data were highly consistent, further confirming at the protein molecular weight level that the engineered bacteria successfully achieved recombinant expression and efficient secretion of human lactoferrin.

[0058] 4) Biological growth kinetics test of engineered strains

[0059] Growth curve determination: To assess whether the expression of recombinant plasmids and exogenous proteins caused a metabolic burden on the host bacteria, untransformed blank *E. coli* (E. coli) and recombinant engineered bacteria transformed with the plasmid (E. coli-Plasmid) were inoculated into LB medium and incubated at 37°C and 220 rpm. Bacterial suspensions were extracted every 1 h, and their absorbance (OD) at 600 nm was measured using a spectrophotometer. 600 ), continuously monitor for 7 hours.

[0060] Results Analysis: For example Figure 4As shown, the growth trends of the engineered bacteria (E. coli-Plasmid) after transformation with the expression plasmid highly overlapped with those of wild-type / blank E. coli during the lag phase, logarithmic growth phase, and plateau phase, with OD600 values ​​steadily increasing to 1.1–1.3 within 7 hours. These results demonstrate that the introduction of the recombinant plasmid and the secretory expression of HLF did not significantly inhibit the physiological activity and proliferation capacity of the bacteria, ensuring the colonization stability for subsequent in vitro and in vivo applications.

[0061] Example 2. Detection of in vitro cellular immune activation and damage-associated molecules (DAMPs) / ferroptosis

[0062] 1) Detection of dendritic cell (DC) phenotype activation and antigen presentation capacity

[0063] BMDC induction and co-incubation: Bone marrow cells were extracted from C57BL / 6 mice and cultured for 6 days in RPMI-1640 complete medium containing GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) to obtain immature bone marrow-derived dendritic cells (BMDCs). Bacterial or cell supernatants from each group (control group, blank E. coli group, and recombinant engineered E. coli (plasmid) group) were co-incubated with BMDCs for 24 h.

[0064] Flow cytometry analysis: DC cells were collected and incubated for 30 min in the dark with fluorescently labeled anti-CD11c, CD80, CD86, and MHC II monoclonal antibodies, respectively. After filtration, the cells were analyzed by flow cytometry.

[0065] See results Figure 5 , Molecular expression ( Figure 5 a): The control group and the blank E. coli group induced The proportion of mature DCs was around 22%; however, under E. coli (plasmid) stimulation, the proportion of mature DCs was significantly increased to over 36% (p < 0.01).

[0066] Molecular expression ( Figure 5 (b) Control group and blank E. coli group The positive rate was approximately 2.2%; however, after treatment with E. coli (plasmid), the positive rate significantly increased to approximately 6% (p < 0.01). This indicates that the HLF-binding bacterial components expressed and secreted by the engineered bacteria can significantly promote the maturation of dendritic cells (DCs) and enhance their antigen-presenting ability.

[0067] 2) Intracellular reactive oxygen species (ROS) accumulation and oxidative stress fluorescence imaging

[0068] Experimental Methods: MC38 colorectal cancer cells were seeded onto slides and divided into a control group, a group treated solely with engineered E. coli (plasmid), and a group treated with engineered bacteria combined with iron (E. coli (plasmid) + iron). The cells were incubated for 12 h. The culture medium was discarded, and 10 μM of DCFH-DA reactive oxygen species (ROS) fluorescent probe was added. The cells were incubated at 37 °C in the dark for 30 min, followed by washing three times with PBS to remove any probes that had not entered the cells. The cell nuclei were then stained with Hoechst 33342, and the intensity of green fluorescence (ROS) was observed using a confocal laser scanning microscope (CLSM).

[0069] Results Analysis: For example Figure 6 As shown, only very weak background fluorescence was observed in the cells of the control group and the single engineered bacteria group; while in the “E. coli(plasmid) + iron” group, strong and dense green fluorescence signals were observed in the cells, confirming that the engineered bacteria can effectively drive intracellular iron accumulation in an iron-rich environment, induce a violent Fenton reaction and produce a large amount of ROS, triggering severe cellular oxidative stress.

[0070] 3) Detection of immunogenic cell death (ICD) markers and damage-associated molecules (DAMPs) release.

[0071] Caloretin (CRT) surface flipping detection: After treatment, MC38 cells were labeled with anti-CRT primary antibody and fluorescent secondary antibody in an unfixed state. Flow cytometry analysis showed that ( Figure 7 In the control group and the single bacterial group, the CRT positivity rate on the cell membrane surface was only about 0.9%; while in the E. coli (plasmid) + iron group, it significantly increased to about 1.5%. This indicates that significant CRT eversion has occurred.

[0072] High-mobility group box 1 (HMGB1) release assay: Flow cytometry was used to analyze the intracellular residual HMGB1 content. The HMGB1 positivity rate in the control group was approximately 1.15%; while in the E. coli (plasmid) + iron group, the intracellular HMGB1 proportion was significantly reduced to approximately 0.6%. () Figure 7 (b) confirmed that a large amount of HMGB1 was released from the nucleus / cytoplasm into the extracellular microenvironment.

[0073] Extracellular ATP release assay: Cell culture supernatant from each group was collected, and the concentration was determined using a luciferase ATP assay kit. Figure 7 (c) The ATP concentration in the supernatant of the control group and the single bacterial group remained at approximately 2.6 nM; while The ATP concentration in the supernatant of the iron supplement group increased explosively to over 6.5 nM. ).

[0074] Conclusion: CRT eversion, HMGB1 release, and massive ATP secretion directly confirm that E. coli (plasmid) combined with iron can induce strong immunogenic cell death (ICD) and ferroptosis in tumor cells.

[0075] Example 3. In vivo intravenous magnetic resonance imaging (MRI), safety evaluation, and validation of synergistic anti-tumor survival.

[0076] 1) In vivo magnetic resonance imaging (MRI) lesion targeting and enhancement effects

[0077] Experimental Methods: A tumor-bearing model was established in C57BL / 6 mice by subcutaneous / oral inoculation with MC38 tumor cells. When the tumor volume grew to approximately 100 mm³, mice were intravenously injected with saline (control group), blank *E. coli* + oral iron supplementation group, and engineered *E. coli* (plasmid) expressing lactoferrin (or combined with iron supplementation) group. At specific time points after drug administration, mice underwent T1 / T2 weighted scans using a mouse magnetic resonance imaging (MRI) system to acquire cross-sectional and coronal images.

[0078] Results Analysis: For example Figure 8 As shown, the engineered E. coli expressing lactoferrin exhibited excellent tumor-targeting colonization and iron-capturing capabilities in vivo. In MRI images, the intestines and tumor lesion areas of this group of mice showed extremely obvious weighted signal contrast enhancement (significantly increased contrast between bright and dark areas), which was significantly better than the control group and the blank bacterial group, confirming that this recombinant engineered bacterial system has excellent function as a live "therapeutic" nano / bioimaging probe.

[0079] 2) In vivo systemic safety assessment

[0080] Experimental methods: During the 7-day treatment and observation period, mice in each group (control group, blank E. coli + iron group, and E. coli expressing lactoferrin + iron group) were electronically weighed daily, and their mental state, feeding status and behavior were closely monitored.

[0081] Results Analysis: For example Figure 9As shown, during the entire treatment observation period, the average weight of mice in each group remained stable within the range of 18-19 g, with a flat curve and no statistical difference. No toxic reactions such as sharp weight loss, diarrhea, or abnormal fur loosening occurred, indicating that the combination therapy of live engineered bacteria and iron supplements has excellent systemic biocompatibility and low toxicity in vivo.

[0082] 3) Analysis of long-term survival rate and tumor-suppressing effect in tumor-bearing mice

[0083] Survival tracking: The long-term survival status of mice in each treatment group (n=6 per group) was monitored, the time of death of mice was recorded, and Kaplan-Meier survival curves were plotted.

[0084] Results Analysis: For example Figure 10 As shown: Control group: Tumors grew rapidly, and the median survival of mice was only 21.5 days, with all mice dying within 26 days; Blank E. coli + iron group: Limited inhibition of tumor growth, with a median survival of 22.5 days, showing no significant improvement compared to the control group; Lactoferrin-expressing E. coli + iron group: Exhibited an extremely efficient in vivo synergistic anti-tumor effect, successfully inhibiting tumor growth, significantly extending the median survival of tumor-bearing mice to 52 days, significantly improving the long-term survival rate of tumor-bearing animals, and achieving excellent synergistic tumor treatment effect.

[0085] In summary, this invention utilizes engineered probiotics to continuously express lactoferrin in situ, establishing a stable iron recruitment system at the tumor site. Combined with iron-rich preparations to continuously replenish iron ions, a persistent local iron reservoir is formed, achieving long-term induction of ferroptosis and enhanced anti-tumor immunity. Simultaneously, local iron enrichment can produce a significant magnetic resonance imaging contrast effect, providing real-time, non-invasive dynamic monitoring of the treatment process.

[0086] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A tumor diagnostic and treatment reagent kit, characterized in that, The kit includes engineered bacteria and an enteric iron source preparation; the engineered bacteria are genetically modified to secrete and express lactoferrin.

2. The reagent kit according to claim 1, characterized in that, The engineered bacteria's chassis bacteria are selected from strains with tumor-targeting ability, intestinal colonization ability, or barrier penetration ability.

3. The reagent kit according to claim 1, characterized in that, The bacteria in the substrate are selected from attenuated Salmonella, Bacillus subtilis, Escherichia coli, lactic acid bacteria, Bifidobacterium, Clostridium, and Listeria; preferably, the bacteria in the substrate are selected from Escherichia coli Nissle1917 (EcN) or attenuated Salmonella VNP20009.

4. The reagent kit according to claim 1, characterized in that, In addition to expressing lactoferrin, the engineered bacteria also co-express an adjuvant therapeutic protein. Preferably, the adjuvant therapeutic protein is selected from one or more of cytokines, immunomodulatory proteins, chemokines, antibodies, nanobodies, or enzymes. More preferably, the adjuvant therapeutic protein is selected from one or more of interferon-γ (IFN-γ), interleukin-12 (IL-12), interleukin-15 (IL-15), PD-L1 nanobody, CTLA-4 nanobody, and glucose oxidase.

5. The reagent kit according to claim 1, characterized in that, The enteric-coated iron source preparation is selected from iron salts or iron chelates coated with an enteric coating; preferably, the iron salt or iron chelate is selected from one or more of ferrous gluconate, ferrous succinate, ferrous fumarate, sodium iron EDTA, iron dextran, and ferric ammonium citrate.

6. A method for preparing a tumor diagnostic and therapeutic reagent kit, characterized in that, The method includes: (1) Construct a recombinant plasmid containing a lactoferrin expression cassette and transform it into a chassis host bacterium to screen for recombinant engineered bacteria that can stably secrete and express lactoferrin; (2) The recombinant engineered bacteria are combined with the enteric iron source preparation to obtain the product.

7. The method for preparing the reagent kit according to claim 6, characterized in that, The method further includes coating the iron source with an enteric material to obtain an enteric iron source formulation; preferably, the recombinant engineered bacteria also co-express the aforementioned adjuvant therapeutic protein of the present invention.

8. The use of the tumor diagnostic kit according to claim 1 in the preparation of medicaments for the prevention, treatment or diagnosis of tumors.

9. The application according to claim 8, characterized in that, The tumor is a solid tumor; preferably, it is colorectal cancer, gastric cancer, pancreatic cancer, or liver cancer; more preferably, it is colorectal cancer.

10. A method for diagnosing and treating tumors, characterized in that, The method includes administering the tumor diagnostic kit of claim 1 to a subject.

Images