Gastric cancer metastatic lymph node detection marker, double-target nano fluorescent probe and preparation method and application thereof

Abstract

The application discloses CD151 gene and ITGAV gene or expression proteins thereof as biomarkers for detection of each stage or the whole stage of lymph node metastasis of gastric cancer in vivo. The application also discloses a CD151 / ITGAV double-target nano-fluorescent probe and a corresponding kit. The CD151 / ITGAV double-target nano-fluorescent probe is prepared from DPPC, cholesterol and DSPE-PEG-NHS as liposome materials, unmodified nano-fluorescent liposomes prepared from the fluorescent agent, and CD151 antibody and ITGAV antibody. The two targets of CD151 and ITGAV are used in the detection of metastatic lymph nodes of gastric cancer. The CD151 / ITGAV double-target nano-fluorescent probe has good complementarity in the whole stage detection of lymph node metastasis of gastric cancer in vivo, and can realize specific and accurate detection of the whole stage of lymph node metastasis of gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the detection of genes and proteins, particularly to markers for detecting metastatic lymph nodes in gastric cancer, dual-target fluorescent nanoprobes, their preparation methods, and applications. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors, and approximately 80%-90% of gastric cancer patients are diagnosed at an advanced stage, resulting in poor prognosis and a heavy disease burden. Lymph node metastasis is the most common form of metastasis in gastric cancer. The Japanese guidelines for the treatment of gastric cancer stipulate that advanced gastric cancer requires dissection of regional drainage lymph nodes (D2). However, due to the lack of intuitive and precise methods for identifying metastatic lymph nodes, this dissection procedure relies heavily on the surgeon's experience. Furthermore, the complex vascular anatomy, regional lymphatic drainage, and tumor biological behavior make precise dissection of metastatic lymph nodes difficult to guarantee: insufficient dissection leaves residual metastatic lymph nodes as a source of postoperative recurrence and metastasis; excessive dissection increases surgical trauma and complications, and weakens the effectiveness of postoperative adjuvant immunotherapy. Therefore, traditional extensive regional drainage lymph node dissection no longer aligns with the principles of minimally invasive and function-preserving surgery. Achieving real-time, precise intraoperative navigation and dissection of metastatic lymph nodes has become crucial for improving the efficacy of radical gastrectomy for gastric cancer.

[0003] To address this challenge, many researchers have modified ICG fluorescent probes for metastatic lymph node imaging, such as through targeted modification and nanomaterial encapsulation. However, most of these modifications are single-target strategies targeting tumor cells. These strategies are often based on the understanding that metastatic lymph nodes are equivalent to the primary tumor, often ignoring the heterogeneity between the primary and metastatic lesions and the complexity of lymph node metastasis in real clinical scenarios. Tumor lymph node metastasis is a complex, multi-stage process. In the same patient, the lymph nodes in the tumor drainage area may include normal lymph nodes, tumor-microinvasive lymph nodes, partially invasive lymph nodes, and completely invasive lymph nodes, even inflammatory lymph nodes after immunotherapy. The staged changes in cell subsets and spatial structure are the root cause of this heterogeneity. In our study, we found that microinvasive lymph nodes have very little tumor infiltration, but are accompanied by a large number of tumor-associated macrophages infiltrating the periphery of the lymph node parenchyma. In this case, conventional tumor-targeting strategies may produce false negative results because they cannot amplify the signal of metastatic lymph nodes. Therefore, screening for specific targets covering different stages of metastasis based on the evolution of cell subpopulations and spatial structures in the microenvironment of metastatic lymph nodes in gastric cancer is an important prerequisite for achieving accurate identification of metastatic lymph nodes.

[0004] In recent years, a tracking technique involving the injection of fluorescent agents around tumors, drainage to regional lymph nodes, and near-infrared II (NIR-II) imaging has shown promise for real-time intraoperative lymph node navigation. However, the development of clinical-grade fluorescent probes for NIR-II window fluorescence imaging is relatively lagging. To address this issue, current mainstream approaches include: 1) developing novel NIR-II fluorescent agents. For example, some researchers have reported a fluorescent agent based on tumor-specific β-galactosidase (β-Gal) for imaging metastatic lymph nodes in gastric cancer (ACS Nano. 2024 Dec 31;18(52):35490-35506.). However, the biocompatibility and in vivo toxicity of such fluorescent agents have not been fully validated clinically, significantly increasing the cost of clinical translation; 2) modifying existing clinically used fluorescent agents. Currently, the most commonly used lymph node probe in clinical practice is indocyanine green (ICG). Although ICG has good biocompatibility and its emission wavelength covers the NIR-II window, making it suitable for intraoperative imaging, it still has limitations such as a short in vivo half-life and lack of tumor targeting. To address this problem, many scholars have modified ICG fluorescent probes for imaging metastatic lymph nodes. For example, one scholar developed an RGD-modified ICG nanoprobe for fluorescence imaging of breast cancer (Adv Sci(Weinh). 2022 Apr;9(12):e2104728). However, most of these modifications are single-target strategies targeting tumor cells, usually based on the understanding that metastatic lymph nodes are equivalent to primary tumors, often ignoring the heterogeneity between primary and metastatic lesions and the complexity of lymph node metastasis in real clinical scenarios. Lymph node metastasis of tumors is a complex, multi-stage process. In the same patient, the lymph nodes draining from the tumor may include normal lymph nodes, microinvasive lymph nodes, partially invasive lymph nodes, and completely invasive lymph nodes, even inflammatory lymph nodes following immunotherapy. We have found that microinvasive lymph nodes show very little tumor infiltration, but are accompanied by a significant number of tumor-associated macrophages infiltrating the periphery of the lymph node parenchyma. In this case, conventional tumor-targeting strategies may produce false-negative results because they cannot amplify the signal from metastatic lymph nodes. Therefore, it is essential to develop biomarkers and reagents for in vivo detection of metastatic lymph nodes in gastric cancer. Finding biomarkers that can detect metastatic lymph nodes at all stages or all stages of gastric cancer is both a key focus and a significant challenge. Summary of the Invention

[0005] Based on this, the purpose of this invention is to cover two biomarkers for specific targets of different lymph node metastasis stages of gastric cancer, their combined use, as well as the preparation of nanofluorescent probes for these specific targets, their preparation methods and applications, so as to achieve accurate identification of metastatic lymph nodes of gastric cancer.

[0006] In a first aspect, the present invention provides the application of CD151 gene and ITGAV gene, or their expressed proteins, in the preparation of a detection kit for all stages or stages of gastric cancer lymph node metastasis; or the application of reagents for detecting the expression levels of CD151 gene and ITGAV gene in the preparation of a detection kit for gastric cancer lymph node metastasis.

[0007] Secondly, the present invention provides a CD151 / ITGAV dual-target fluorescent nanoprobe, which is prepared by making a fluorescent nanoliposome from liposome material and fluorescent agent, and then labeling it with an antibody, wherein the antibody is a D151 antibody and an ITGAV antibody.

[0008] In some embodiments, the nanofluorescent liposomes are prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS, and a fluorescent agent in a molar ratio of 28-32:25-30:2-4:3-5.

[0009] In some embodiments, the fluorescent agent is ICG-COOH.

[0010] Optionally, the molar ratio of the CD151 antibody and ITGAV antibody to DSPE-PEG-NHS in the nanofluorescent liposomes is 1:1:1400-2100, more preferably 1:1:1400-1600.

[0011] Thirdly, the present invention provides the application of the CD151 / ITGAV dual-target fluorescent nanoprobe described in the second aspect in the preparation of a gastric cancer lymph node metastasis detection kit.

[0012] Fourthly, the present invention provides a full-stage detection kit for all stages of gastric cancer lymph node metastasis, wherein the kit includes reagents for detecting the expression levels of CD151 gene and ITGAV gene in vivo.

[0013] The reagents include various existing RNA sequencing methods, RT-qPCR, and Western blot, immunofluorescence, flow cytometry, or immunohistochemistry using antibodies with relevant functions such as CD151 and ITGAV.

[0014] In some embodiments, the reagent comprises the CD151 / ITGAV dual-target fluorescent nanoprobe described in the second aspect.

[0015] In some implementations, the detection is performed inside the subject.

[0016] In some implementations, the detection is targeted at all or all stages of lymph node metastasis in gastric cancer.

[0017] In some implementations, the subject is a mammal; for example, the term "subject" or "patient" refers to a human or a non-human animal such as a mammal. "Subject" can include any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. Human subjects can be referred to as patients.

[0018] Fifthly, the present invention provides a method for preparing a CD151 / ITGAV dual-target fluorescent nanoprobe, which includes the following steps:

[0019] Unmodified fluorescent nanoliposomes were prepared by using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG-NHS as liposome materials and a fluorescent agent.

[0020] The fluorescent nanoliposomes were reacted with CD151 antibody and ITGAV antibody in the dark.

[0021] The unreacted NHS groups are quenched and then ultrafiltered to obtain the final product.

[0022] In some embodiments, the molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS, and the fluorescent agent is 28-32:25-30:2-4:3-5; and / or the molar ratio of the CD151 antibody and ITGAV antibody to the DSPE-PEG-NHS in the unmodified fluorescent nanoliposomes is 1:1:1400-2100, more preferably 1:1:1400-1600.

[0023] In a sixth aspect, the present invention provides the application of the CD151 gene or its expressed protein in the preparation of a gastric cancer lymph node metastasis detection kit; or the application of a reagent for detecting the expression level of the CD151 gene in the preparation of a gastric cancer lymph node metastasis detection kit.

[0024] In a seventh aspect, the present invention also provides a CD151 target fluorescent nanoprobe, which is prepared by making a fluorescent nanoliposome from liposome material and a fluorescent agent, and then labeling it with a CD151 antibody.

[0025] Preferably, the nanofluorescent liposomes are prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS, and a fluorescent agent in a molar ratio of 28-32:25-30:2-4:3-5.

[0026] Preferably, the molar ratio of the CD151 antibody to the unmodified fluorescent nanoliposomes containing DSPE-PEG-NHS is 1:1400-2100, more preferably 1:1400-1800.

[0027] Eighthly, the present invention also provides the application of the CD151 target nanofluorescent probe described in the seventh aspect in the preparation of a gastric cancer lymph node metastasis detection kit.

[0028] This invention discovers the specificity of two targets, CD151 and ITGAV, in the metastatic lymph node microenvironment and applies them to the detection of metastatic lymph nodes in gastric cancer, particularly suitable for in vivo detection. Moreover, the two targets, CD151 and ITGAV, have excellent complementarity in the detection of all stages of gastric cancer lymph node metastasis in vivo, enabling specific and accurate detection of all stages of gastric cancer lymph node metastasis.

[0029] Furthermore, a dual-target fluorescent nanoprobe—NP-αCD151 / αITGAV@ICG—was prepared, exhibiting excellent targeting capabilities. It can encompass the specific drug targets CD151 and ITGAV within the lymph node microenvironment at different metastatic stages, demonstrating excellent in vivo tracing effects on metastatic lymph nodes at all stages. This invention amplifies the fluorescence signal by targeting the specific targets CD151 and ITGAV within the metastatic lymph node microenvironment, providing a novel approach to fluorescence imaging of metastatic lymph nodes. Attached Figure Description

[0030] Figure 1 Transcriptome and single-cell sequencing clarified the characteristics of changes in cell subsets in metastatic lymph nodes of gastric cancer and the screening of specific targets; (A) differential gene analysis and specific target screening process between normal lymph nodes and metastatic lymph nodes; (B) tSNE diagram and cell subset ratio stacking diagram of normal lymph nodes and metastatic lymph nodes; (C) expression of CD151 and ITGAV in cell subsets.

[0031] Figure 2 Immunohistochemistry and multilabel fluorescence were used to preliminarily verify the expression and colocalization of target points in the lymph node microenvironment at different metastatic stages; (A) Schematic diagram of lymph node metastasis stages; (B) Immunohistochemical detection of CD151 and ITGAV protein expression in lymph nodes at different metastatic stages (scale bar: 100 μm); (C) Statistical graph of immunohistochemistry.

[0032] Figure 3 In vitro cell co-culture experiments were conducted to verify the effect of gastric cancer tumor cells on the expression of CD151 and ITGAV in target cells; (A) RT-qPCR was used to detect the transcriptional level of CD151 and ITGAV genes in target cells after co-culture; (B) Flow cytometry was used to detect the protein expression level of CD151 and ITGAV in target cells after co-culture.

[0033] Figure 4The morphology and particle size distribution of the synthesized nanofluorescent probes in this invention; (A) NP-αCD151 / αITGAV@ICG (G4), NP-αCD151@ICG (G3), NP-αITGAV@ICG (G2), NP@ICG (G1), schematic diagram of the nanofluorescent probes under transmission electron microscopy (scale bar: 100nm); (B) DLS shows the particle size distribution of each group of nanofluorescent probes.

[0034] Figure 5 The effects of different modifications on the targeting of fluorescent nanoprobes; (A) confocal microscopy to observe the uptake of different fluorescent nanoprobes by different cells (scale bar: 50 μm); (B) flow cytometry to quantitatively detect the differences in uptake rates of different fluorescent nanoprobes by different cells.

[0035] Figure 6 : In vivo targeting verification of nanofluorescent probes; (A) in vivo imaging observation of fluorescence intensity of different fluorescent probes in metastatic lymph node regions; (B) comparison of fluorescence intensity between metastatic lymph node and normal lymph node regions; (C) comparison of fluorescence intensity difference between metastatic lymph node and normal lymph node regions for different fluorescent probes.

[0036] Figure 7 : Validation of the efficacy and safety of nanofluorescent probes for identifying metastatic lymph nodes in nude mice; (A) ROC curves of different fluorescent probes for identifying metastatic lymph nodes in nude mice in vivo imaging; (B) Pathological HE of different fluorescent probes corresponding to important organs in nude mice.

[0037] Figure 8 : Validation of the recognition efficacy of dual-targeted P-αCD151 / αITGAV@ICG nanofluorescent probes for metastatic lymph nodes in New Zealand white rabbits; (A) Schematic diagram of New Zealand white rabbit processing procedure; (B) Typical in vivo schematic diagram of lymph node tracing by different fluorescent probes; (C) In vitro fluorescence of different fluorescent probes and corresponding pathological schematic diagrams; (D) ROC curves of different fluorescent probes for metastatic lymph node recognition; (E) Schematic diagram of the safety test results of important organs of New Zealand white rabbits using NP-αCD151 / αITGAV@ICG.

[0038] Figure 9 Verification of the recognition efficacy of nano-fluorescent probes for metastatic lymph nodes in human ex vivo gastric cancer specimens; (A) Schematic diagram of human ex vivo gastric cancer specimen processing flow; (B) Typical schematic diagram of intraoperative probe tracing of lymph nodes in human ex vivo specimens; (C, D) ROC curves of NP-αCD151 / αITGAV@ICG nano-fluorescent probes for metastatic lymph node recognition; (E) ROC curves of NP-αITGAV@ICG nano-fluorescent probes for metastatic lymph node recognition; (F) ROC curves of NP-αCD151@ICG nano-fluorescent probes for metastatic lymph node recognition; (G) Schematic diagrams of lymph node HE and pan-CK.

[0039] Figure 10 Multi-label fluorescence detection of CD151 and ITGAV expression and cellular sublocalization in lymph nodes at different metastatic stages, including the colocalization of the two targets ITGAV and CD151 with CD68-positive monocytes / macrophages and SMA-positive fibroblasts in metastatic lymph nodes at each stage (scale bar: 100 μm).

[0040] Figure 11 The pathological hematologic reaction (HE) of rabbit micrometastatic lymph nodes m-MLN and partially metastatic p-MLN, and the targeting distribution of the dual-target fluorescent nanoprobe NP-αCD151 / αITGAV@ICG on cells within lymph nodes.

[0041] Figure 12 The pathological hematologic reaction (HE) of human micrometastatic lymph nodes (m-MLN) and partially metastatic p-MLN, and the targeting distribution of the dual-target fluorescent nanoprobe NP-αCD151 / αITGAV@ICG on cells within lymph nodes. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0045] The ITGAV (Integrin Subunit Alpha V) gene is located on the long arm of human chromosome 2 at 2q32.1, CD51, VNRA (Vitronectin Receptor Alpha), and MSK8, encoding the integrin αV subunit. It forms various RGD sequence-specific receptors by pairing with different β subunits (such as β1, β3, β6, etc.), playing a central role in cell adhesion, signal transduction, angiogenesis, and TGF-β activation.

[0046] The CD151 gene, also known as the RAPH blood group antigen or TSPAN24, is a protein encoded by the CD151 gene in humans. It belongs to the Transmembrane 4 Superfamily (TM4SF), also known as the tetraspanin family. The CD151 gene is a key molecule connecting the extracellular matrix and intracellular signaling networks, playing a dual role in maintaining normal tissue structure and promoting tumor metastasis.

[0047] Some embodiments of the present invention provide a comprehensive targeted nanofluorescent probe for metastatic lymph nodes in all stages of gastric cancer. The nanofluorescent probe is prepared by mixing a monoclonal antibody targeting the selected target, a liposome raw material, and an ICG fluorescent probe.

[0048] In some implementations, we screened for specific targets in metastatic lymph nodes of gastric cancer at different stages: We collected transcriptome sequencing data of lymph nodes from cancer patients in public databases for differential gene analysis, screening for membrane protein targets for which corresponding antibodies have been applied in clinical trials (CD151 and ITGAV were identified). We further collected single-cell sequencing data of gastric cancer lymph nodes from public databases, generated a matrix, and performed quality control, standardization, and data integration. We then analyzed the changes in the proportions of T cells, B cells, macrophages, fibroblasts, and tumor cells in normal-micrometastatic-partially metastatic-completely metastatic lymph nodes as tumor invasion progressed, as well as the expression of CD151 and ITGAV in different cell subpopulations. We collected lymph node samples from gastric cancer surgeries, stained them with hematoxylin and eosin (HE) to clarify tumor infiltration, and preliminarily verified the spatiotemporal evolution of the microenvironment by comparing the spatial structure, cell morphology, and type of the invaded and normal parts of lymph nodes in pathological sections at different metastatic stages. Further, IHC staining was used to statistically analyze the differences in average optical density (IOD / Area) of target sites such as CD151 and ITGAV in the stained areas of metastatic and normal lymph nodes. Multi-label immunofluorescence staining (CD68, α-SMA, CD151, ITGAV) was used to observe the expression and co-localization of CD151 and ITGAV, verifying the complementarity of the two target sites. The co-localization of CD151 and ITGAV with monocytes, macrophages, and fibroblasts was observed to clarify the specificity of the two target sites CD151 and ITGAV in the microenvironment of metastatic lymph nodes. Human leukemia monocyte cell line (THP-1), THP-1 M0 macrophages induced by phorbol ester (PMA), and fibroblasts (NF-8) were co-cultured with gastric cancer cell line (NUGC-3). RNA and cells were collected at different time points for RT-qPCR and flow cytometry to observe the expression of ITGAV and CD151 in metastatic lymph node-related cells after co-culture. (The RT-qPCR amplification system is as follows: 1. RNA extraction: Using the Takara RNA reverse transcription kit, 1 μg of RNA was de-DNase-free and reverse transcribed according to its instructions to obtain 20 μL of cDNA; 2. Amplification: Amplification was performed using the Novozymes enzyme system (product number Q712-02). The main components were 10 μL of SYBR enzyme, 0.1 μL of Forward Primer, 0.1 μL of Reverse Primer, 1 μL of the above cDNA, and 8.2 μL of ddH2O.)

[0049]

[0050] (2) Preparation of dual-target fluorescent nanoprobes:

[0051] DPPC, cholesterol, and DSPE-PEG-NHS were added to a mixed reaction system with a molar ratio of DSPC, cholesterol, and DSPE-PEG-NHS of 30:27:3. Liposomes were labeled with ICG-COOH as a fluorescent agent. The lipid mixture was dissolved in an organic solvent, rotary evaporated to form a thin film, vacuum dried, hydrated with HEPES solution at 50°C, sonicated, and extruded through a 200 nm filter membrane to form unmodified fluorescent nanoprobes (NP@ICG). αITGAV and αCD151 were added to the liposome suspension at pH 8.0 and 4°C with a molar ratio of antibody to DSPE-PEG-NHS of 1:1:1500. The reaction was stirred in the dark for 2 hours. The reaction was terminated by adding 0.2 mM glycine. The mixture was then subjected to a 100 kDa ultrafiltration tube to remove free antibody molecules and DSPE-PEG-NHS, followed by sucrose density gradient centrifugation to remove uncoupled liposomes. The fluorescent nanoprobe NP-αCD151 / αITGAV@ICG was successfully synthesized.

[0052] The present invention will be further described in detail below with reference to specific embodiments.

[0053] Example 1: Spatiotemporal evolution of the lymph node microenvironment in gastric cancer and screening of specific targets

[0054] Volcano maps of gene expression in lymph nodes were obtained by analyzing public databases (GSE6605, GSE44408, GSE60542, GSE95109, GSE26571, GSE2665, GSE28442). Figure 1 A) identified 22 membrane protein targets that are specific to metastatic lymph nodes and for which targeted drugs have been applied clinically. Figure 1 A), and further validation was performed using single-cell transcriptome sequencing data from metastatic and normal lymph nodes in clinical samples (GSE190870, GSE188807, GSE189042, GSE163558, GSE282542, GSE167036, GSE184362, GSE199619), to analyze the changes in cell subsets of lymph nodes at different metastatic stages (). Figure 1B): Normal lymph nodes are mainly composed of T and B cells, with very few monocytes, macrophages, and fibroblasts. Micrometastatic lymph nodes are accompanied by a small number of tumor cells, and the number of macrophages and monocytes increases significantly. As tumor cells proliferate and grow, T and B cells gradually deplete, while the number of macrophages and monocytes further increases, and fibroblasts begin to appear and increase significantly, but the number of tumor cells only increases slightly. As T and B cells are almost exhausted, tumor cells proliferate rapidly, the number of monocytes and macrophages decreases, and fibroblasts coexist with tumor cells. At this point, tumor cells completely infiltrate and occupy the lymph nodes. Further repeated validation of the 22 selected genes in tumor cells, tumor-associated monocytes, macrophages, and fibroblasts revealed that ITGAV and CD151 are highly expressed in tumor cells, tumor-associated monocytes, macrophages, and fibroblasts, respectively, but almost not expressed in T and B cells of normal lymph nodes. Their expression in monocytes, macrophages, and fibroblasts of normal lymph nodes is significantly lower than that in metastatic lymph node-associated cells. Figure 1 C).

[0055] Example 2

[0056] Twenty-nine metastatic lymph node samples were collected from seven patients with gastric cancer, and 15 normal lymph nodes were collected from two patients with gastric cancer and three patients with colorectal cancer (the distinction between metastasis and normal lymph nodes was mainly based on the immunohistochemical staining of pan-cytoketarin in metastatic lymph nodes being ++ or higher; if pathological results were available, they were also considered). Formalin was fixed for 24 hours, paraffin-embedded, and sections were prepared. Immunohistochemical (IHC) experiments were performed using CD151 and ITGAV antibodies. The expression differences of the two target genes, CD151 and ITGAV, in metastatic and normal lymph nodes were observed. It was found that both CD151 and ITGAV genes were highly expressed in metastatic lymph nodes, significantly higher than in normal lymph nodes, and the area of ​​high expression increased with increasing metastasis. Figure 2(A, B, C). IF results showed that the expression and cellular sublocalization of CD151 and ITGAV in metastatic lymph nodes of gastric cancer exhibited stage-like changes: almost no expression in normal lymph nodes (NLN); minimal tumor cell infiltration in micrometastatic lymph nodes (m-MLN), but a large number of tumor-associated macrophages (TAMs) highly expressing CD151 infiltrating locally; significant tumor cell infiltration in partial-metastatic lymph nodes (p-MLN), accompanied by TAMs and fibroblasts highly expressing CD151 and ITGAV; and predominantly CD151-expressing tumor cells in complete-metastatic lymph nodes (c-MLN), accompanied by a large number of CD68 cells highly expressing CD151 and ITGAV. + TAMs and α-SMA + Fibroblast infiltration ( Figure 10 ).

[0057] Example 3

[0058] This embodiment verifies the results through in vitro experiments. Human mononuclear cell lines THP-1 and M0 macrophages (THP-1+PMA induced) were co-cultured with the human gastric cancer cell line NUGC-3. After 24 hours of indirect co-culture, RT-qPCR revealed that the mRNA transcription levels of CD151 and ITGAV genes were increased. Figure 3 CD11b was screened by flow cytometry after direct co-culture for 48 hours (A). + Cellular analysis revealed that, compared to the blank control group, the proportion of THP-1 and M0 macrophage monocyte lines positive for both ITGAV and CD151 was significantly increased (referring to THP-1 and M0 macrophage monocyte lines stained with flow cytometry antibodies containing CD151 and ITGAV fluorescent groups, respectively, compared to THP-1 and M0 macrophage monocyte lines not stained with flow cytometry antibodies containing CD151 and ITGAV fluorescent groups). Figure 3 In a study (B), human fibroblast cell line NF-8 and human gastric cancer cell line NUGC-3 were co-cultured indirectly for 12 hours. RT-qPCR revealed increased mRNA transcription levels of CD151 and ITGAV genes. Figure 3 (A) Direct co-culture for 24 hours followed by flow cytometry screening of α-SMA +After cell analysis, it was found that the proportion of ITGAV and CD151 double-positive cells (specifically referring to human NF-8 fibroblast cells stained with flow cytometry antibodies containing CD151 and ITGAV fluorescent groups, showing the fluorescence of the flow cytometry antibody compared to human NF-8 fibroblast cells stained with flow cytometry antibodies without CD151 and ITGAV fluorescent groups) in the human fibroblast cell line NF-8 was significantly increased compared to the blank control group. Figure 3 (B)

[0059] 2. Construction, characterization and in vitro evaluation of nanofluorescent probes.

[0060] Dipalmitoylphosphatidylcholine (DPPC, purchased from Anaiji Company, catalog number M0108535-63-89-8), cholesterol (Chol, purchased from Aladdin, catalog number 57-88-5), and DSPE-PEG-NHS (purchased from Chongqing Yusi Pharmaceutical, catalog number 1445723-73-8) were used as the main components of liposomes, and ICG-COOH (purchased from Xinweichuang Biotechnology Company, catalog number RJWZ2386 ICG-COOH) was used as the fluorescent agent. The molar ratio of DPPC:Chol:DSPE-PEG-NHS:ICG-COOH was 30:27:3:4. Unmodified fluorescent nanoliposomes were prepared by thin-film dispersion using conventional methods, and the dispersion was carried out by ultrasonication. Subsequently, the unmodified fluorescent nanoliposomes were repeatedly extruded through a 200 nm polycarbonate membrane to obtain unmodified fluorescent nanoliposomes. Subsequently, under pH 8.0 HEPES solution conditions, ITGAV antibody (αITGAV, Intetumumab, purchased from Tauss Biosciences, catalog number T76812, CAS No. 725735-28-4) and CD151 antibody (αCD151, purchased from Leinco, catalog number C2039, clone number 50-6) were reacted with the NHS groups on the surface of liposomes in the dark for 2 hours (each antibody was added to unmodified fluorescent nanoliposomes (DSPE-PEG-NHS) at a molar ratio of 1:1500, i.e., NP-αCD151 / αITGAV@ICG was αCD151:αITGAV:DSPE-PEG-NHS=1:1:1500, NP-αCD151@ICG was αCD151:DSPE-PEG-NHS=1:1500, NP-αITGAV@ICG was αITGAV The ratio of DSPE-PEG-NHS was 1:1500, and unreacted NHS groups were quenched with 0.2 mM glycine. The reaction system was then ultrafiltered using a 100 kDa ultrafiltration tube to successfully synthesize the nano-fluorescent probe NP-αCD151 / αITGAV@ICG. Following the same method and process, targeted modified nano-fluorescent probes (NP-αCD151 / αITGAV@ICG (G4), NP-αITGAV@ICG (G3), NP-αCD151@ICG (G2), and NP@ICG (G1)) were synthesized sequentially. DLS particle size analysis of the nano-fluorescent probes revealed an average particle size of 100-130 nm, and a uniform spherical morphology under TEM. Figure 4 (A) Weekly DLS particle size analysis of the above materials revealed good stability. Figure 4 (B) Encapsulation rate is 75%-82%.

[0061] In vitro, 10 μM nanofluorescent probes NP-αCD151 / αITGAV@ICG, NP-αCD151@ICG, NP-αITGAV@ICG, and NP@ICG were co-incubated for two hours with various target cells associated with metastatic lymph nodes of gastric cancer (gastric cancer cell line NUGC-3, and three cell lines directly co-cultured with NUGC-3: THP-1, M0 macrophages, and NF-8). The results were analyzed using a fluorescence microscope. Figure 5 (A) and flow cytometry ( Figure 5 (B) Verification showed that among the nanofluorescent probes, NP-αCD151 / αITGAV@ICG exhibited the best targeting for each cell type.

[0062] 3. Preclinical evaluation of the safety and effectiveness of nanofluorescent probes in identifying metastatic lymph nodes at all stages using a nude mouse model.

[0063] A BALB / c nude mouse model of popliteal lymph node metastasis was established, divided into five groups: NP-αCD151 / αITGAV@ICG (group G5), NP-αCD151@ICG (group G4), NP-αITGAV@ICG (group G3), NP@ICG (group G2), and free-ICG (group G1), with 5-6 mice in each group. NP-αCD151 / αITGAV@ICG, NP-αCD151@ICG, NP-αITGAV@ICG, NP@ICG, and free-ICG (ICG-COOH 20 nmol / mouse) were administered subcutaneously to the paw pads around the tumor and on the normal side. One hour after administration, in vivo imaging revealed that the fluorescence intensity in the metastatic lymph node region of the targeted modification groups (G3-5) was significantly higher than that in the normal lymph node region. Figure 6 (AB), among which the fluorescence intensity difference between metastatic lymph nodes and normal lymph nodes in the NP-αCD151 / αITGAV@ICG group was significantly higher than that in other groups ( ). Figure 6 C). Metastatic lymph nodes and paired normal lymph nodes from the contralateral side were dissected. ROC curves were plotted based on fluorescence intensity. Comparison of AUC values ​​revealed that NP-αCD151 / αITGAV@ICG had the highest AUC value (0.9348), exhibiting the highest AUC value for identifying metastatic lymph nodes, indicating the strongest recognition ability. Specificity and sensitivity analysis at different thresholds of the ROC curve showed 84.78% specificity and 89.58% sensitivity. Similarly, NP-αCD151@ICG had an AUC value of 0.7517, with a specificity of 74.07% and a sensitivity of 70.37%. NP-αITGAV@ICG had an AUC value of 0.8957, with 82.61% specificity and 90.00% sensitivity. Figure 7A). Simultaneously, in vivo observation of other lymph node metastasis and tracing revealed that the NP-αCD151 / αITGAV@ICG, NP-αCD151@ICG, NP-αITGAV@ICG, NP@ICG, and ICG groups all showed good safety in the heart, liver, spleen, lungs, kidneys, and intestines of nude mice. Figure 7 B). The results showed that NP-αCD151@ICG and NP-αITGAV@ICG had good targeting of metastatic lymph nodes in vivo. However, the nanofluorescent probe (NP-αCD151 / αITGAV@ICG), which was modified with both aCD151 and aITGAV antibodies, had better targeting of metastatic lymph nodes in vivo compared with only one antibody or no antibody modification.

[0064] Example 4: Preclinical evaluation of the safety and efficacy of the nanofluorescent probe in identifying metastatic lymph nodes using a New Zealand white rabbit model.

[0065] A New Zealand white rabbit gastric cancer lymph node metastasis model was constructed using conventional methods. Figure 8 (A) The tumor was divided into two groups: NP-αCD151 / αITGAV@ICG and free-ICG, with 3 animals in each group. NP-αCD151 / αITGAV@ICG and free-ICG (ICG-COOH 300um, 500ul / point) were administered at four points around the tumor. 0.5 hours after administration, in vivo imaging showed that the fluorescence intensity in the metastatic lymph node area of ​​the targeted modification group (NP-αCD151 / αITGAV@ICG) was significantly higher than that in the normal lymph node area. (Diagram showing intraoperative fluorescence laparoscopic lymph node fluorescence tracing of metastatic lymph nodes.) Figure 8 B). In the dual-target modified group, metastatic lymph nodes and normal lymph nodes were well distinguished due to the significant difference in fluorescence intensity, while in the ordinary ICG group, the fluorescence intensity of normal lymph nodes was also significantly increased, making them indistinguishable from metastatic lymph nodes. Figure 8 C). Metastatic and normal lymph nodes were dissected, and ROC curves were plotted based on fluorescence intensity. It was observed that NP-αCD151 / αITGAV@ICG showed the strongest ability to identify metastatic lymph nodes, with an AUC value of 0.9233. Specificity and sensitivity analysis at different thresholds of the ROC curve revealed an achievable specificity of 83.3% and a sensitivity of 92.5% at the inflection point of the ROC curve. Figure 8 D).

[0066] We also tested the safety of NP-αCD151 / αITGAV@ICG in vital organs of rabbits and found that the safety was good. Figure 8 E).

[0067] Simultaneously, we analyzed rabbit micrometastatic lymph nodes (m-MLN) and partially metastatic p-MLN, demonstrating the corresponding pathological hematologic reaction (HE) and the targeted distribution of the dual-target fluorescent nanoprobe NP-αCD151 / αITGAV@ICG on lymph node cells. It was observed that NP-αCD151 / αITGAV@ICG was well taken up by cells within both micrometastatic and partially metastatic lymph nodes, indicating that it targets and is taken up by cells within metastatic lymph nodes, thus demonstrating good targeting activity against both micrometastatic and partially metastatic lymph nodes. Figure 11 ).

[0068] Example 5: Preclinical validation of the safety and effectiveness of nanofluorescent probes in identifying metastatic lymph nodes using a human ex vivo gastric cancer specimen model.

[0069] In Example 3, NP-αCD151 / αITGAV@ICG, NP-αITGAV@ICG, and NP-αCD151@ICG (ICG-COOH 400µm, 300µl / point) were administered to excised specimens from gastric cancer patients at four points around the tumor. Figure 9 A), 0.5 hours after drug administration, in vivo imaging revealed that the fluorescence intensity of the metastatic lymph node region in the targeted modification group (NP-αCD151 / αITGAV@ICG) was significantly higher than that in the normal lymph node region, indicating that the metastatic lymph nodes and normal lymph nodes in the dual-target modification group were well distinguished due to the significant difference in fluorescence intensity. Figure 9 B). Metastatic lymph nodes and normal lymph nodes were dissected and identified based on absolute fluorescence intensity (B). Figure 9 C) and the presence or absence of fluorescence intensity visible to the naked eye ( Figure 9 D) ROC curve plotting revealed that NP-αCD151 / αITGAV@ICG has a very strong ability to identify metastatic lymph nodes, with AUC values ​​of 0.903 and 0.895, respectively. The method of distinguishing metastatic and non-metastatic lymph nodes based on the presence or absence of fluorescence by the naked eye, which best matches the clinical intraoperative findings, can achieve a specificity of 83.8% and a sensitivity of 95.2%.

[0070] Similarly, NP-αITGAV@ICG has a specificity of 80.00% and a sensitivity of 63.16%. Figure 9 E). NP-αCD151@ICG has a specificity of 63.89% and a sensitivity of 70.00% (E). Figure 9 F). Our criterion for determining whether lymph node metastasis has occurred is whether pan-CK staining is positive (F). Figure 9 G).

[0071] Simultaneously, we detected and analyzed human micrometastatic lymph nodes (m-MLN) and partially metastatic p-MLN using the above method; the results are shown below. Figure 12The results showed that the corresponding pathological HE and the targeted distribution of the dual-target nanofluorescent probe NP-αCD151 / αITGAV@ICG on lymph node cells were well observed. It can be seen that NP-αCD151 / αITGAV@ICG was well taken up by micrometastatic lymph nodes and some metastatic lymph node cells. It is achieved by targeting and taking up cells in metastatic lymph nodes, and has a good targeting effect on micrometastatic and some metastatic lymph nodes.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of CD151 gene and ITGAV gene, or their expressed proteins, in the preparation of a detection kit for all stages or stages of gastric cancer lymph node metastasis; or the application of reagents for detecting the expression levels of CD151 gene and ITGAV gene in the preparation of a detection kit for all stages or stages of gastric cancer lymph node metastasis.

2. A CD151 / ITGAV dual-target fluorescent nanoprobe, characterized in that, It is prepared by making nano-fluorescent liposomes from liposome materials and fluorescent agents, and then labeling them with antibodies, namely CD151 antibody and ITGAV antibody.

3. The CD151 / ITGAV dual-target fluorescent nanoprobe according to claim 2, characterized in that, The nanofluorescent liposomes are prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS, and fluorescent agents in a molar ratio of 28-32:25-30:2-4:3-5.

4. The CD151 / ITGAV dual-target fluorescent nanoprobe according to claim 3, characterized in that, The fluorescent agent is ICG-COOH.

5. The CD151 / ITGAV dual-target fluorescent nanoprobe according to any one of claims 3-4, characterized in that, The molar ratios of the CD151 antibody and ITGAV antibody to the DSPE-PEG-NHS in the nanofluorescent liposomes were 1:1:1400-2100, respectively.

6. The application of the CD151 / ITGAV dual-target fluorescent nanoprobe according to any one of claims 2-5 in the preparation of a gastric cancer lymph node metastasis detection kit; preferably, the detection is for each or all stages of gastric cancer lymph node metastasis.

7. A full-stage detection kit for all stages of gastric cancer lymph node metastasis, characterized in that, The kit includes reagents for detecting the expression levels of the CD151 and ITGAV genes, preferably, the reagents can be detected in the subject.

8. The kit according to claim 6, characterized in that, The reagent includes the CD151 / ITGAV dual-target fluorescent nanoprobe as described in any one of claims 2-5.

9. A method for preparing a CD151 / ITGAV dual-target fluorescent nanoprobe, comprising the following steps: Unmodified fluorescent nanoliposomes were prepared by using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and DSPE-PEG-NHS as liposome materials and a fluorescent agent. The fluorescent nanoliposomes were reacted with CD151 antibody and ITGAV antibody in the dark. The unreacted NHS groups are quenched and then ultrafiltered to obtain the final product.

10. The preparation method according to claim 9, characterized in that, The molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS, and fluorescent agent is 28-32:25-30:2-4:3-5; and / or the molar ratio of CD151 antibody and ITGAV antibody to DSPE-PEG-NHS in unmodified fluorescent nanoliposomes is 1:1:1400-2100.

11. The application of the CD151 gene or its expressed protein in the preparation of a gastric cancer lymph node metastasis detection kit; or the application of reagents for detecting CD151 gene expression in the preparation of a gastric cancer lymph node metastasis detection kit.

12. A CD151 target nanofluorescent probe, characterized in that, It is prepared by making nano-fluorescent liposomes from liposome materials and fluorescent agents, and then labeling them with CD151 antibodies; preferably, the nano-fluorescent liposomes are prepared by dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), DSPE-PEG-NHS and fluorescent agents in a molar ratio of 28-32:25-30:2-4:3-5.

13. Application of the CD151 target nanofluorescent probe in the preparation of a gastric cancer lymph node metastasis detection kit according to claim 12.

Images