Tissue transparentizing method and immunostaining method for esophagus of multi-stage canceration mouse

Abstract

The invention discloses a tissue transparentizing method and an immunostaining method for esophagus of a multi-stage canceration mouse. The tissue transparentizing method comprises a whole tissue immunostaining step, a transparentizing step, a three-dimensional imaging step, a histological staging step and an image analysis step, wherein the whole tissue immunostaining step comprises the step of carrying out whole tissue immunostaining on a mouse esophagus sample by adopting an antibody; and a histological staging step, including de-transparentizing the three-dimensional imaging sample, and then performing pathological staining to judge the pathological staging of the sample. According to the application, a mouse esophageal whole tissue immunostaining method and a post-transparentizing esophageal original tissue pathological staging method are researched and developed for the first time, and a basis is provided for researching three-dimensional space characteristics of multi-stage lesions of mouse esophageal squamous carcinoma; the invention constructs a set of tissue transparentizing technology flow to realize microenvironment three-dimensional characteristic analysis in the mouse esophageal squamous cell carcinoma progress process.

Description

Technical Field

[0001] This application relates to the field of tissue transparency technology, and in particular to a tissue transparency method and immunostaining method for the esophagus of mice at multiple stages of cancer. Background Technology

[0002] Tumor progression is dynamically regulated in the spatiotemporal environment of the tissue (including immune cells and blood vessels). However, the three-dimensional spatial characteristics of vascular remodeling or immune microenvironment changes during the entire carcinogenesis process of a tumor have not yet been elucidated. This is partly due to the difficulty in obtaining continuous tissue samples from multiple disease stages of human tumors, and partly due to limitations in three-dimensional imaging technology.

[0003] Because obtaining multi-stage pathological tissues from patients to illustrate the entire carcinogenesis process is challenging, a mouse esophageal squamous cell carcinoma orthotopic model induced by 4-nitroquinoline-1-oxide (4NQO) has become an important tool for reproducing the pathological process of human esophageal squamous cell carcinoma and studying tumor progression. The application of single-cell transcriptome sequencing technology in this model has further deepened our understanding of the cellular dynamics and gene expression changes in the esophageal squamous cell carcinoma microenvironment, confirming its role in understanding the changes in cellular and gene expression during the development and progression of esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is a malignant tumor originating from the squamous epithelium of the mucosa, exhibiting a typical multi-stage pathological process of carcinogenesis, including stages from normal, inflammation, hyperplasia, dysplasia, carcinoma in situ, to invasive carcinoma.

[0004] Tissue transparency technology is an emerging three-dimensional imaging technique involving a series of steps: tissue fixation and pretreatment, endogenous or exogenous fluorescent labeling, sample dehydration and defatting to achieve transparency, refractive index matching, microscopic imaging, and data processing. Currently, this technology has been successfully applied to analyze the three-dimensional spatial structural features of various solid tumors.

[0005] However, there are currently no studies applying tissue transparency technology to three-dimensional imaging of esophageal squamous cell carcinoma lesions at multiple stages. Furthermore, the differences in tissue characteristics, the difficulty of whole-tissue antibody labeling, and the challenges of identifying the original pathological stage significantly hinder research on the progression of esophageal squamous cell carcinoma.

[0006] Therefore, when using a mouse esophageal squamous cell carcinoma orthotopic model to study the pathological process and tumor evolution of human esophageal squamous cell carcinoma, how to solve the above problems, namely, differences in tissue characteristics, difficulties in whole-tissue antibody labeling, and how to identify the original pathological stage, and effectively achieve tissue transparency, remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this application is to provide an improved method for tissue clearing and immunostaining of the esophagus in mice with multiple stages of cancer.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] One aspect of this application discloses a method for tissue clearing of mouse esophagus at multiple stages of cancer, including whole-tissue immunostaining, clearing, three-dimensional imaging, histological staging, and image analysis. The whole-tissue immunostaining step includes whole-tissue immunolabeling of mouse esophageal samples using selected antibodies; the clearing step includes clearing the immunolabeled mouse esophageal samples; the three-dimensional imaging step includes three-dimensional imaging of the cleared mouse esophageal samples; the histological staging step includes de-clearing the three-dimensionally imaged mouse esophageal samples, paraffin embedding, sectioning, and pathological staining of the de-cleared samples, and determining the pathological stage of the samples based on the pathological staining results; the image analysis step includes analyzing the three-dimensional images of tissues at the same pathological stage using analysis software and performing intergroup comparison analysis. The analysis software includes, for example, Imaris or Fiji software; the pathological staining includes, for example, H&E staining.

[0010] It should be noted that this application creatively proposes histological staging of transparent mouse esophageal samples after three-dimensional imaging, and proposes analyzing three-dimensional images of tissues at the same pathological stage and performing intergroup comparison analysis, thereby obtaining a complete transparency process applicable to multi-stage mouse esophageal lesion research. The tissue transparency method for multi-stage carcinomatous mouse esophagus in this application provides a new scheme and approach for the study of multi-stage squamous cell carcinoma lesions in mouse esophagus. In this application, multi-stage carcinomatous mouse esophagus mainly refers to multi-stage squamous cell carcinoma esophageal lesions in mice.

[0011] It should also be noted that the histological staging in this application can refer to existing pathological staging. However, this application's research found that there exists a pre-inflammatory phase between the normal phase and the inflammatory phase, i.e., before the typical inflammatory phase. Although it is difficult to distinguish between the pre-inflammatory phase and the normal phase pathologically, vascular remodeling and T-cell infiltration changes have already occurred in the pre-inflammatory phase, which can be distinguished using the tissue transparency process of this application. Therefore, in one implementation of this application, the pathological staging is creatively divided into the normal phase (NOR), pre-inflammation (PINF), inflammatory phase (INF), dysplasia (DYS), and invasive carcinoma (ICA).

[0012] In one implementation of this application, the antibody used in the whole-tissue immunostaining step is rat anti-CD31 and / or rabbit anti-CD3.

[0013] It should be noted that screening suitable antibodies for immunostaining is an important step for successful whole-tissue immunolabeling. This study found that rat anti-CD31 and rabbit anti-CD3 antibodies can meet the requirements for whole-tissue immunostaining of mouse esophagus, laying the foundation for the subsequent tissue transparency process of whole-tissue mouse esophagus.

[0014] In one implementation of this application, the whole-tissue immunostaining step involves whole-tissue immunolabeling of mouse esophageal samples, specifically including one of the following two methods.

[0015] 1) Passive Wild DISCO immunostaining protocol: The mouse esophageal sample to be stained is incubated in Wild DISCO permeation blocking solution, and then passively incubated in Wild DISCO immunostaining solution containing primary antibody. After incubation, it is washed with PBS. Then, the mouse esophageal sample is incubated in Wild DISCO immunostaining solution containing secondary antibody. After incubation, it is washed with PBS. This completes the staining of the entire mouse esophageal tissue.

[0016] 2) iDISCO centrifugation staining protocol: Pretreated mouse esophageal samples to be stained were incubated in iDISCO permeation buffer and iDISCO blocking buffer, respectively. Then, the mouse esophageal samples were incubated in iDISCO primary antibody incubation buffer containing primary antibody. During incubation, the samples were centrifuged at 400-600g for 4-6 hours daily, while the antibody concentration of the primary antibody was gradually increased until the incubation in iDISCO primary antibody incubation buffer was completed. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature. Then, the mouse esophageal samples were incubated in iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI. Similarly, the samples were centrifuged at 400-600g for 4-6 hours daily. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature, thus completing the staining of the entire mouse esophageal tissue.

[0017] The pretreatment can be performed using either a non-methanol pretreatment method or a methanol pretreatment method, depending on whether the screened antibody is compatible with methanol. For specific methanol and non-methanol pretreatment methods, please refer to the existing iDISCO staining protocol.

[0018] In one implementation of this application, the passive Wild DISCO immunostaining protocol involves incubation for 1-2 days in 4-6 mL of Wild DISCO permeation blocking solution, incubation for 7-10 days in 1.5-2 mL of Wild DISCO immunostaining solution containing primary antibody, and incubation for 7-10 days in 1.5-2 mL of Wild DISCO immunostaining solution containing secondary antibody. After primary antibody incubation, the PBS washing method is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the PBS washing method is 10-20 mL for 2-2.5 days, 10-12 times.

[0019] In one implementation of this application, the iDISCO centrifugal staining protocol involves incubation for 1.5-2 days in 4-6 mL of iDISCO permeation buffer, incubation for 2-3 days in 4-6 mL of iDISCO blocking buffer, incubation for 3.5-4.5 days in 1.5-2 mL of iDISCO primary antibody incubation buffer containing primary antibody, and incubation for 3.5-4.5 days in 1.5-2 mL of iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI. After primary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 10-12 times.

[0020] In one implementation of this application, the histological staging step specifically includes: immersing the transparent mouse esophageal sample sequentially in an aqueous methanol solution with gradually decreasing concentration, then washing it overnight in PBS to achieve detransparentization; then, placing the detransparent sample sequentially in an aqueous ethanol solution with gradually increasing concentration for dehydration, treating it with xylene, and then impregnating it with paraffin.

[0021] In one implementation of this application, the methanol aqueous solution with gradually decreasing concentration in the histological staging step is 100% methanol, 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, and 20% methanol aqueous solution.

[0022] In one implementation of this application, the method of soaking in methanol aqueous solution with gradually decreasing concentration is as follows: first, soaking in 100% methanol at least twice, each time using 4-6 mL of 100% methanol for 1-1.2 hours; then, soaking in 4-6 mL of 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, and 20% methanol aqueous solution for 1-1.2 hours respectively.

[0023] In one implementation of this application, after soaking in a methanol aqueous solution, the sample is washed overnight in 15-50 mL of PBS.

[0024] In one implementation of this application, the ethanol aqueous solution with gradually increasing concentration is 75% ethanol aqueous solution, 85% ethanol aqueous solution, 90% ethanol aqueous solution, 95% ethanol aqueous solution, and 100% ethanol.

[0025] In one implementation of this application, the dehydration is carried out gradually in ethanol aqueous solutions with progressively increasing concentrations by sequentially soaking the sample in 75% ethanol aqueous solution, 85% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution for 40-60 minutes each, and then soaking it in 100% ethanol at least twice, each time for 30-40 minutes.

[0026] In one implementation of this application, the xylene treatment includes immersion in xylene at least twice, each time for 20-30 minutes.

[0027] It should be noted that one of the key aspects of this application is the subsequent de-transparency of the mouse esophagus after 3D imaging, followed by histological staging, i.e., histopathological staging. This is something that existing mouse esophageal research techniques lack. This application is the first to propose and implement a scheme for histopathological staging of samples after 3D imaging, and proposes to analyze the 3D images of tissues at the same pathological stage based on the pathological staging results and perform intergroup comparative analysis. Understandably, the paraffin embedding, sectioning, and pathological staining of the samples can all refer to existing techniques, such as conventional H&E staining.

[0028] Another aspect of this application discloses an immunostaining method for mouse esophagus at multiple stages of carcinogenesis, comprising performing whole-tissue immunostaining on mouse esophageal samples using rat anti-CD31 and / or rabbit anti-CD3.

[0029] It should be noted that the immunostaining method of this application is actually the whole-tissue immunostaining step in the tissue transparency process of the multi-stage carcinogenic mouse esophagus of this application; therefore, its specific schemes, including the passive wildDISCO immunostaining scheme and the iDISCO centrifugation staining scheme, refer to the tissue transparency process of this application, and will not be repeated here.

[0030] Another aspect of this application discloses the use of an antibody in preparing a tissue clearing kit for mouse esophagus at multiple stages of carcinogenesis, wherein the antibody is rat anti-CD31 and / or rabbit anti-CD3.

[0031] It should be noted that one of the key aspects of this application is the development of two antibodies that can meet the requirements for whole-tissue immunostaining of mouse esophagus, and the development of a complete set of tissue transparency methods for mouse esophagus in multiple stages of cancer. Therefore, this application actually discovers a new use for two antibodies, rat anti-CD31 and rabbit anti-CD3, namely, that they can be used to prepare tissue transparency kits for mouse esophagus in multiple stages of cancer, and used as antibodies for whole-tissue immunostaining in the tissue transparency process, thereby laying the foundation for the tissue transparency process of mouse esophagus in multiple stages of cancer.

[0032] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0033] This application pioneered the development of a whole-tissue immunostaining method for mouse esophagus and a pathological staging method for the original esophageal tissue after transparency, providing a foundation for studying the three-dimensional spatial characteristics of multi-stage lesions of mouse esophageal squamous cell carcinoma; this application also creatively constructed a set of tissue transparency technology procedures to achieve three-dimensional characteristic analysis of the microenvironment during the progression of mouse esophageal squamous cell carcinoma. Attached Figure Description

[0034] Figure 1 These are images of mouse esophageal squamous cell carcinoma tissues under different transparency conditions in the embodiments of this application;

[0035] Figure 2 This is a diagram showing the results of antibody verification and methanol compatibility in the embodiments of this application;

[0036] Figure 3 These are the results of two whole-tissue immunostaining protocols in the embodiments of this application;

[0037] Figure 4 This is a photograph of tissue in the invasive carcinoma stage after transparent imaging and H&E staining in an embodiment of this application.

[0038] Figure 5 This is a flowchart of the whole-tissue transparency technique for the carcinogenesis process of mouse esophageal squamous cell carcinoma in the embodiments of this application;

[0039] Figure 6 This is a diagram of mouse esophageal tissue samples and corresponding pathological stages in an embodiment of this application;

[0040] Figure 7 These are three-dimensional images of esophageal blood vessels at different pathological stages in the embodiments of this application;

[0041] Figure 8 This is a result diagram of the imaging depth of the light sheet microscope in the embodiments of this application;

[0042] Figure 9 This is a computational modeling diagram of blood vessels in an embodiment of this application;

[0043] Figure 10These are vascular analysis diagrams of different pathological stages of esophageal squamous cell carcinoma in the embodiments of this application;

[0044] Figure 11 This is a diagram showing the changes in the vascular ring structure and straightness in the embodiments of this application;

[0045] Figure 12 This is a spatiotemporal dynamic distribution diagram of T cells and T cell aggregates in the embodiments of this application;

[0046] Figure 13 This is a graph showing the quantitative analysis results of T cells in an embodiment of this application. Detailed Implementation

[0047] The present application will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained from the description in the specification and general technical knowledge in the art.

[0048] Example

[0049] I. Main Materials and Equipment

[0050] 1. Laboratory animals

[0051] Six-week-old female C57BL / 6 mice (The Jackson Laboratory) were housed in an environment free of specific pathogens, provided with standard feed and bedding, allowed free access to food, and kept at an ambient temperature of 20-22°C with a 12-hour day-night cycle.

[0052] 2. Experimental reagents

[0053] 4% paraformaldehyde (Soluble), PBS (pH 7.4) (Gibco), dichloromethane (Sigma), dibenzyl ether (Sigma), methanol (Sangon Biotech), ethanol (Sangon Biotech), Triton X-100 (Sigma), donkey serum (Jackson Immuno Research Laboratories), BSA (Beyotime), heparin (Sangon Biotech), CHAPS (Sangon Biotech), NP-40 (Beyotime), glycine (Sangon Biotech), DMSO (Soluble), sodium deoxycholate (Sangon Biotech), hepta(2,6-di-O-methyl)-β-cyclodextrin (Lexin), Tween-20 (Sangon Biotech), FDISCO ex vivo clearing kit (Giavis), CUBIC-L / R+(M) kit (TCI), rat anti-mouse CD31 antibody (Clone MEC 13.3 (RUO)) (BD) Biosciences), Rabbit anti-CD3 antibody (E4T1B) (CST), Recombinant rabbit anti-CD4 antibody (EPR19514) (Abcam), Rabbit anti-F4 / 80 antibody (D4C8V) (CST), Donkey anti-rabbit IgG, Alexa488 (Invitrogen), Donkey anti-rabbit IgG, Alexa 647 (Invitrogen), Donkey anti-rabbit IgG, Alexa 555 (Abcam), DAPI (Sigma), Sodium azide (Sigma), 4NQO (Sigma), 1,2-propanediol (Sigma), Xylene (Sangon).

[0054] 3. Experimental Apparatus

[0055] Medical refrigerators (Aucma), medical freezers (Panasonic), small high-speed centrifuges (Eppendorf), low-speed centrifuges (Ausun), heating and drying ovens (Thermo). Scientific, paraffin embedding machine (Leica), paraffin microtome (Leica), clean bench (Sujing Antai), panoramic slide scanning system VS200 (Olympus), vortex shaker (Scientific Industries), pipette (Rainin), electric pipette (Brand), slide staining and mounting workstation (Leica), cryostat (Leica), ice maker (Panasonic), ultrapure water treatment system (Milli-Q), double-door chromatography freezer (Zhixin), weakly corrosive fireproof safety cabinet (Jinming), flammable liquid fireproof safety cabinet (Jinming), rotary table (Qilinbell), biochemical incubator (Yiheng), Sterllaris confocal microscope (Leica), rotating confocal microscope (Andor), light slide microscope (Litone), peristaltic pump (Lange), shaker (Qilinbell), fume hood (De'an), electronic analytical balance (METTLER TOLEDO).

[0056] 4. Experimental Consumables

[0057] Pipette tips (Rainin), pipettes (10mL, 25mL) (Corning), Pasteur pipettes (Biosharp), centrifuge tubes (15mL, 50mL) (Corning), EP tubes (1.5mL, 2mL, 5mL) (Biosharp), culture dishes (Corning), sterile syringes (i-Quip), disposable sterile injection needles (Kejin), 0.22μm filters (Millipore), blue-capped glass bottles (100mL, 1L) (Shuniu), 5mL glass vials (VWR), forceps, microscissors, tissue scissors, hemostatic forceps (Reward), confocal dishes (Biosharp), adhesive slides (Shitai), tissue embedding cassettes (Shitai), disposable latex gloves (Kejin), activated carbon masks (Maidikang), scalpel blades (Leica), coverslips (Shitai).

[0058] 5. Preparation of main solutions

[0059] (1) Cardiac perfusion irrigation fluid (500 mL)

[0060] 500 mL of pH 7.4 PBS, 0.033 g of heparin.

[0061] Weigh out heparin and dissolve it in 0.01M PBS at pH 7.4 to a final concentration of 10 U / mL. After shaking to dissolve, store at 4°C.

[0062] (2) WildDISCO permeation sealing solution (100mL)

[0063] Triton X-100 2mL, donkey serum 10mL, PBS pH 7.4 88mL, sodium azide 0.02g.

[0064] Using a pipette and balance, add the above reagents to 88 mL of 0.01 M PBS, shake to dissolve, and then sterilize with a filter. Prepare fresh before use.

[0065] (3) WildDISCO immunostaining solution (100mL)

[0066] Donkey serum 3 mL, Triton X-100 2 mL, CHAPS 10 g, glycine 1 g, DMSO 10 mL, hepta(2,6-di-O-methyl)-β-cyclodextrin 1 g, PBS pH 7.4 85 mL, sodium azide 0.02 g.

[0067] Take the corresponding dose of the above reagents and add them to 85 mL of 0.01 M PBS. Shake to dissolve and then sterilize with a filter. Prepare fresh each time you use it.

[0068] (4) PTx.2 solution (500 mL)

[0069] 500 mL of pH 7.4 PBS and 1 mL of Triton X-100.

[0070] Add 1 mL of Triton X-100 to 500 mL of 0.01 M PBS, shake to dissolve, sterilize with a filter, and store at room temperature.

[0071] (5) PTwH solution (500mL)

[0072] 500 mL of pH 7.4 PBS, 1 mL of Tween-20, and 0.005 g of heparin.

[0073] Take the corresponding dose of the above reagents and add them to 500 mL of 0.01 M PBS. Shake to dissolve, then sterilize with a filter and store at room temperature.

[0074] (6) iDISCO pretreatment solution 1 (500 mL)

[0075] 400 mL of pH 7.4 PBS, 1 mL of Triton X-100, and 100 mL of DMSO.

[0076] Add the corresponding doses of the above reagents to 400 mL of 0.01 M PBS, shake to dissolve, filter to sterilize, and store at room temperature.

[0077] (7) iDISCO pretreatment solution II (100mL)

[0078] 80 mL of pH 7.4 PBS, 100 μL of Triton X-100, 100 μL of Tween-20, 0.1 g of sodium deoxycholate, 100 μL of NP-40, and 20 mL of DMSO.

[0079] Add the corresponding doses of the above reagents to 80 mL of PBS, shake to dissolve, and then sterilize with a filter. Prepare fresh each time you use it.

[0080] (8) iDISCO osmotic buffer (50 mL)

[0081] PTx.2 40mL, glycine 1.15g, DMSO 10mL, sodium azide 0.01g.

[0082] Take the corresponding dose of the above reagents and add them to 40 mL of PTx.2. Shake to dissolve, then sterilize with a filter and store at room temperature.

[0083] (9) iDISCO blocking solution (50mL)

[0084] PTx.2 42mL, donkey serum 3mL, DMSO 5mL, sodium azide 0.01g.

[0085] Take the corresponding dose of the above reagents and add them to 42 mL of PTx.2. Shake to dissolve, then sterilize with a filter. Prepare fresh each time you use it.

[0086] (10) iDISCO primary antibody incubation solution (20 mL)

[0087] PTwH 18.4mL, DMSO 1mL, donkey serum 600μL, sodium azide 4mg.

[0088] Take the corresponding dose of the above reagents and add them to 18.4 mL of PTwH. Shake to dissolve, then sterilize with a filter. Prepare fresh each time you use it.

[0089] (11) iDISCO secondary antibody incubation solution (20 mL)

[0090] PTwH 19.4 mL, donkey serum 600 μL, sodium azide 4 mg.

[0091] Take the corresponding dose of the above reagents and add them to 19.4 mL of PTwH. Shake to dissolve, then sterilize with a filter. Prepare fresh each time you use it.

[0092] (12) 4NQO stock solution (40mL)

[0093] 40 mL of 1,2-propanediol, 0.2 g of 4NQO.

[0094] Weigh 0.2g of 4NQO powder using a balance and add it to 40mL of 1,2-propanediol. Shake and dissolve overnight at 4°C, then store at 4°C protected from light.

[0095] II. Experimental Methods

[0096] 1. Animal modeling and material selection

[0097] Six-week-old female wild-type C57BL / 6 mice had their drinking water replaced with 200 mL of 4NQO solution. This was achieved by mixing 4NQO stock solution and sterile water at a 1:50 ratio to obtain a final concentration of 100 μg / mL. Mice were allowed free access to the 4NQO solution, which was then replaced with normal sterile water. The 4NQO solution was refreshed weekly. Heart perfusion was performed at 0, 6, 13, 17, and 25 weeks after the mice had free access to the 4NQO solution. After perfusion, the esophagus was longitudinally dissected to facilitate mucosal staining. The tissue was then laid flat on paper and post-fixed to prevent morphological alteration.

[0098] 2. Cardiac perfusion esophageal tract

[0099] (1) Intraperitoneal injection of 150 μL of 1% sodium pentobarbital to anesthetize mice to a deep anesthetized state. After anesthesia, fix the mice on a foam board in a fume hood. Use curved tissue scissors to cut open the skin of the mouse's abdominal and thoracic cavities, then cut the diaphragm and the ribs on both sides. Use hemostats to flip up and fix the anterior ribs to expose the heart. Be careful not to accidentally cut large blood vessels and organs.

[0100] (2) After the right atrial appendage is cut open with microscissors, the butterfly needle connected to the peristaltic pump tube is immediately inserted into the left apex of the heart. At a rate of less than 10 mL / min, about 50 mL of cardiac perfusion flushing fluid is injected until the liver turns light brown. Then the tube is placed into a conical flask containing 4% paraformaldehyde and about 40 mL of paraformaldehyde is injected at the same rate until the mouse body becomes rigid.

[0101] (3) After the perfusion was completed, the mouse esophagus was quickly dissected and cut from the tongue down to the stomach. The tissue was then soaked in PBS containing 0.02% sodium azide for trimming. The esophagus was then cut longitudinally and laid flat on a small piece of A4 paper. The paper was soaked in 5 mL of 4% paraformaldehyde and fixed overnight at 4°C.

[0102] (4) The fixed tissue was washed three times with PBS for one hour each time. The washed tissue could be stored at 4°C for several weeks in PBS solution containing 0.02% sodium azide.

[0103] 3. Verification of antibody properties and methanol compatibility

[0104] Determining whether an antibody is suitable for whole-tissue immunostaining is a crucial step for successful three-dimensional staining. Before staining, it is necessary to verify whether the screened antibody can penetrate thick sections. Additionally, the iDISCO pretreatment step requires testing the antibody's methanol compatibility. Therefore, this experiment used thick frozen sections (20 μm) for verification. The sections were first incubated in 100% methanol for 3 hours, while untreated sections served as a positive control.

[0105] After fixation and washing, the tissues were dehydrated in 20% and 30% sucrose double-distilled aqueous solutions, respectively, until the tissues sank from the top to the bottom of a 50 mL tube. The tissues were then embedded in OCT using a cryostat and rapidly cooled to -22°C. Sections were then prepared at a thickness of 20 μm, and the intact sections were adhered to glass slides and stored at -20°C. Before staining, the sections were allowed to stand at room temperature for 30 minutes, incubated with methanol, permeabilized with 0.4% Triton X-100 for 30 minutes, and then blocked with 5% donkey serum and 2% BSA for 1 hour. The sections were then incubated at 4°C for 20 hours in 2.5% donkey serum / 1% BSA / PBS containing primary antibody. After washing five times for 5 minutes each time, the sections were incubated with secondary antibody in PBS at room temperature for 1.5 hours. After washing five times for 5 minutes each time, the nuclear cells were stained with DAPI. The sections were imaged under a confocal microscope. If the antibody can effectively penetrate thick frozen sections, it can be used for whole-tissue immunostaining, and we will screen for its optimal incubation concentration. Additionally, if methanol pretreatment reduces the signal-to-noise ratio, the methanol-free iDISCO sample pretreatment method will be used.

[0106] 4. Whole-tissue immunomarking protocol

[0107] Whole-tissue immunolabeling is a crucial step in three-dimensional imaging and is often a key factor leading to experimental failure. Here, we designed two protocols suitable for antibody labeling of mouse esophagus: the passive WildDISCO protocol, primarily derived from the WildDISCO staining protocol, but to reduce antibody usage, we did not use the active pump in the WildDISCO protocol, instead utilizing only its staining solution for passive antibody incubation, achieving three-dimensional immunolabeling of mouse esophagus in a more economical way; the iDISCO centrifugation protocol references antibody centrifugation incubation methods from the literature and has been adjusted for applicability, mainly by introducing centrifugation and gradient osmosis during antibody incubation. Details are as follows:

[0108] (1) Passive Wild DISCO Immunostaining Protocol: After fixation and washing, incubate the tissue in 5 mL of Wild DISCO permeation blocking solution for 1.5 days, then incubate the tissue in 1.8 mL of Wild DISCO immunostaining solution containing primary antibody for 7-10 days. Wash the tissue with PBS 8 times over 2 days. Incubate the tissue in 1.8 mL of Wild DISCO immunostaining solution containing secondary antibody for 7-10 days, avoiding light. Wash the tissue with PBS 10 times over 2 days. All steps are performed with gentle rotation at room temperature.

[0109] (2) iDISCO centrifugation protocol: After pretreatment, mouse esophageal tissue was incubated twice for 1 hour in PTx.2 solution, then overnight in iDISCO pretreatment solution one, followed by overnight incubation in iDISCO pretreatment solution two. Finally, the mouse esophageal tissue samples were washed twice for 1 hour in PTx.2 solution. After room temperature pretreatment, the samples were incubated at 37°C for 1.5 days in 5 mL of iDISCO permeation buffer, and then incubated at 37°C for 2 days in blocking buffer. The tissues were then placed in 1.8 mL of iDISCO primary antibody incubation solution containing primary antibodies (CD3, 1:200; CD31, 1:20) and incubated at 37°C for 4 days, centrifuged at 500 g for 5 hours daily, with a gradient increase in antibody concentration (e.g., CD3 concentrations of 1:800, 1:600, 1:400, 1:200). The tissues were washed eight times with PTwH for 2 days at room temperature. The tissue was then incubated in 1.8 mL of iDISCO secondary antibody incubation solution containing secondary antibody and DAPI at 37°C for 4 days, centrifuged at 500 g for 5 hours daily. Finally, the tissue was washed with PTwH for 10 times over 2 days at room temperature. Except for centrifugation, all steps were performed with gentle rotation on a gyroscope. It is worth noting that the centrifuged tissue may become bent and deformed, but this deformation can be reversed by the holder in subsequent light section microscopy.

[0110] 5. Mouse esophageal transparency protocol

[0111] Both of the above whole-tissue immunolabeling protocols are compatible with the iDISCO clearing protocol. Labeled esophageal tissue is placed in a 5mL glass vial and gradually dehydrated for 1 hour in double-distilled aqueous solutions of 20%, 40%, 60%, 80%, and 100% methanol, followed by dehydration in 100% methanol for 2 hours. The tissue is then incubated at room temperature in a 66% dichloromethane / 33% methanol solution for 3 hours, followed by dehydration and defatting twice in 100% dichloromethane for 15 minutes each time. Rotation is required in all these steps, with a speed of less than 15 rpm. Finally, the sample is immersed in a glass vial filled with dibenzyl ether until clear. The cleared tissue is imaged within one week.

[0112] We used the dehydration and degreasing time as an optimization condition, setting dehydration and degreasing times of 0.8, 1, and 1.2 times respectively to compare the final transparency of the tissue.

[0113] 6. Three-dimensional imaging scheme

[0114] Litone light-panel fluorescence microscopy: for esophageal tissue (approximately 8×2×0.7~1.3mm) 3Three-dimensional imaging was performed. To achieve high imaging accuracy, a custom 10x objective lens (Olympus, XLPLN10XSVMP / NA 0.6 / maximum working distance 7.2mm) was used to clearly observe T cells, with a resolution of 2048×2048 pixels and a voxel size of 0.58×0.58×2μm. Multichannel images were acquired using the following fluorescent markers: DAPI, Alexa Fluor 555, and Alexa Fluor 647. The raw images were acquired in 16-bit TIFF format. All images were stitched together into a complete 3D image using the Liscan plugin.

[0115] 7. Histological staging scheme after three-dimensional imaging

[0116] To determine the primary pathological stage of esophageal squamous cell carcinoma progression in the original esophageal tissue, we performed pathological staging of the mouse esophagus using the optimized DIPCO protocol after three-dimensional imaging. The main improvements were shortening the de-transparentification and dehydration embedding conditions to suit transparent esophagus.

[0117] After 3D imaging, the clear tissue was de-cleared by soaking in 5 mL of 100% methanol twice for 1 hour each, and then in 5 mL of 80%, 60%, 40%, and 20% methanol aqueous solutions for 1 hour each. The tissue was then washed overnight in 20 mL of PBS. All steps were performed at room temperature using a gyroscope. Esophageal tissue was dehydrated twice, sequentially immersed in a series of aqueous ethanol solutions (75%, 85%, 90%, 95%) for 40 minutes each, and then in anhydrous ethanol for 30 minutes each. Finally, it was treated twice in xylene for 30 minutes each, followed by paraffin infiltration. The paraffin infiltration temperature was 60°C, and the infiltration time was 50 minutes twice. The longitudinal section of the tissue was placed perpendicular to the embedding cassette. After the paraffin solidified, the embedding cassette and the tissue were stored at -20°C.

[0118] Intermittent sections were prepared from the paraffin-embedded tissue. The section thickness was 3 μm. After 5-10 sections, the next set of sections was prepared at 20 μm intervals. Complete sections were selected and placed in hot water at 42℃ for expansion. The sections were then lifted out with an adhesive slide and baked at 40℃ to remove moisture. Finally, the sections were stored at room temperature. The slides were subjected to routine H&E staining in a slide staining and mounting workstation (i.e., xylene 15 min, xylene 15 min, anhydrous ethanol 3 min, anhydrous ethanol 3 min, 95% ethanol 3 min, 85% ethanol 3 min, 75% ethanol 3 min, double distilled water 3 min, hematoxylin staining solution 3 min, tap water 2 seconds, separation 3 seconds, tap water 15 min for bluing, eosin staining solution 5 min, tap water to wash off eosin, 75% ethanol 10 seconds, 85% ethanol 2 min, 95% ethanol 2 min, anhydrous ethanol 2 min, anhydrous ethanol 2 min, xylene 5 min, xylene 5 min, neutral resin mounting). After staining, the entire slide was scanned using a 20x objective lens of a panoramic slide scanning system, and histopathological examination was performed by pathology students. Our defined histopathological classification includes the NOR stage (i.e., normal stage), where the epithelium shows good stratification. The PINF stage (pre-inflammation) represents the lesion between the normal and inflammatory stages. The INF stage (inflammation) is characterized by lymphocytic infiltration in the epithelium. The DYS stage (dysplasia) is defined as the presence of atypical cells in the epithelium, but not reaching the full thickness. The ICA stage (invasive carcinoma) is defined as cancer cells invading the subepithelial tissue.

[0119] 8. Image Processing and Analysis

[0120] Imaris (version 10.0.0) was primarily used for 3D image processing and analysis. Three mouse esophagi were used for each pathological stage. Three cubic-shaped epithelial or tumor regions were randomly selected from each esophageal tissue for analysis. Considering the different locations of the esophageal epithelium on different XY planes, each cube had a volume of approximately 700 × 250 × 40 μm. 3 Up to 600×300×60μm 3For vessel segmentation, an AI-driven filament module was used to detect CD31-positive signals. An automated path algorithm and manual creation were employed to achieve the best fit. The diameter, volume, area, number of branches, number of branch points, number of endpoints, perfusion resistance, and straightness values ​​were then obtained from Imaris statistics. The average values ​​for vessel diameter, volume, area, perfusion resistance, and straightness at each time point were calculated across all vessels in nine regions. The average value for the relative ring structure of vessels at each time point was calculated as the average of the number of branch points / endpoints across the nine regions. The number of branches at each time point was statistically calculated as the average of the nine regions. For T cell identification, an 8μm diameter spot and the default quality filter were used for rendering. The T cell density was calculated as the number / volume per region. For T cell aggregates, a surface with a diameter of 20-200μm (6×1.2×0.4mm) was calculated for each esophagus. 3 The density is within a certain range. The tumor surface is determined by the surface manual drawing wizard, and the number of T cells at different distances from the tumor surface can be found in the statistics module.

[0121] The quantification of average fluorescence intensity was performed using Fiji. Two-dimensional images were uniformly captured in different XY planes from the outermost to the bottom of the tissue. The outermost image was imported into Fiji, and the signal was selected using the default threshold. The average grayscale value was analyzed. All operations could be saved to the macro recorder and the same analysis could be performed on all subsequent images. Finally, the average grayscale value of all images was normalized using Excel to obtain the intensity of the outermost surface.

[0122] 9. Statistical Analysis

[0123] Statistical analysis was performed using GraphPad Prism 8. The Shapiro-Wilk test was performed to assess normality when n < 50. Differences between groups were determined using a two-tailed unpaired t-test (for normally distributed groups or those with n > 50) or the Mann-Whitney U test (for non-normally distributed groups). Data are expressed as mean ± sem. P < 0.05 was considered statistically significant.

[0124] III. Results and Analysis

[0125] 1. The transparency condition of mouse esophagus

[0126] Good tissue transparency is crucial for 3D imaging. We effectively removed lipids and water by varying the treatment time and dosage of these clearing agents. 5 mL of clearing agent was used in all groups, and dehydration and degreasing times were set at 0.8, 1, and 1.2 times in the iDISCO group. Results are as follows: Figure 1 As shown.

[0127] Figure 1The results showed that iDISCO-0.8x and iDISCO-1x times both achieved good transparency, with the tissue appearing slightly yellow and the bottom grid lines clearly visible. However, the transparency effect was slightly weaker at iDISCO-1.2x times.

[0128] 2. Validation of antibody properties and methanol compatibility

[0129] In whole-tissue immunostaining protocols, it is necessary to verify whether the antibody is suitable for three-dimensional tissue labeling and whether it is compatible with methanol. Considering these two aspects, multiple antibodies for the tissue microenvironment were stained and validated using thick frozen sections. Positive results were ultimately obtained for two antibodies (CD31 and CD3), as shown in Table 1. Figure 2 As shown.

[0130] Table 1 Optimal incubation concentration of antibody

[0131] Antibody Item number concentration rat anti-CD31 550274 1:20 Rabbit anti-CD3 78588 1:200 Donkey anti-rabbit IgG, Alexa 647 A-31573 1:1000 Donkey anti-rat IgG, Alexa 555 ab150154 1:1000 DAPI D9542 1:500

[0132] Figure 2 The image shows the results of immunofluorescence verification of the methanol compatibility of the antibody on a thick frozen section (20 μm). Figure (a) shows the CD3... + (a) Comparison of T cells (red) in the non-methanol pretreatment and methanol pretreatment groups, with blue indicating DAPI nuclear staining; (b) CD31-labeled blood vessels (green).

[0133] Figure 2 The results showed that the rat anti-CD31 and rabbit anti-CD3 antibodies had low signal-to-noise ratios in the methanol group, indicating that non-methanol pretreatment methods should be used.

[0134] 3. Whole-tissue immunomarking protocol

[0135] Whole-tissue immunostaining is a crucial step in three-dimensional imaging and is often a key factor leading to experimental failure. This study established two staining protocols that enable effective antibody penetration of the esophageal epithelium, such as... Figure 3 As shown, both protocols can be combined with iDISCO clearing. In short, the iDISCO-centrifugation protocol enhances antibody penetration through centrifugation and gradient antibody incubation. The passive wildDISCO protocol is an active perfusion method that does not use the original protocol; it only utilizes its wildDISCO staining solution for passive incubation. This optimized protocol is more economical than the original wildDISCO protocol and is feasible for staining single tissues. Figure 3 These are the results of two whole-tissue immunostaining protocols in the embodiments of this application. Red indicates CD31-labeled blood vessels, and blue indicates esophageal epithelial regions representing strong DAPI positive signals. Scale bar: 30 μm.

[0136] 4. Histological staging

[0137] To explore the three-dimensional spatial structure of esophageal squamous cell carcinoma at multiple stages of its development, this study innovatively proposed identifying the pathological stage of the imaged tissue before image analysis. This facilitates the analysis of three-dimensional images of tissues at the same pathological stage and allows for intergroup comparative analysis. Hematoxylin and eosin (H&E) staining is the gold standard for pathological diagnosis. This study referenced and optimized the DIPCO protocol to adapt to the pathological staging of esophageal tissue after imaging, primarily by shortening the tissue dehydration and embedding time to reduce esophageal tissue shrinkage. Furthermore, the final H&E staining pattern allowed us to distinguish the pathological stage of the tissue. Figure 4 As shown. Figure 4 Photograph showing tissue in the invasive carcinoma stage after clear imaging and H&E staining. Scale bar: 50 μm.

[0138] 5. Work Process

[0139] Based on the optimization of the above series of schemes, we finally established a whole-tissue transparency technology process for exploring the carcinogenesis and progression of esophageal squamous cell carcinoma in mice, such as... Figure 5 As shown in the diagram. The workflow is as follows: First, esophageal tissue from mice at different induction stages is perfused and collected. Then, after verifying the relevant antibodies, whole-tissue immunolabeling is performed, using either of the two staining protocols described above. Next, the esophagus is cleared using iDISCO (1x time). Then, three-dimensional imaging is performed using a light-panel microscope, which provides better imaging results. After that, the tissue is de-cleared, paraffin-embedded, and routinely H&E stained to verify the histopathological stage. Finally, three-dimensional analysis of tissues at different stages is performed using Imaris software. Figure 5 The process of tissue transparency technique for multi-stage esophageal lesions in mice is illustrated using Figdraw.

[0140] 6. Establishment of a multi-stage esophageal squamous cell carcinoma lesion model in mice

[0141] We performed tissue clearing experiments on the esophagus of mice at 0, 6, 13, 17, and 25 weeks after drug administration. After three-dimensional imaging, we used the optimized DIPCO protocol for pathological staging. More than 50% of the tissue at each age was in the same pathological stage. We selected three mice from each stage for subsequent analysis. Based on H&E staining results, this study divided the pathological staging into five phases: normal (NOR), pre-inflammation (PINF), inflammation (INF), dysplasia (DYS), and invasive carcinoma (ICA). These 15 mice corresponded to the five pathological stages: normal (NOR), pre-inflammation (PINF), inflammation (INF), dysplasia (DYS), and invasive carcinoma (ICA). Figure 6 As shown. Figure 6 The diagram shows the 4NQO-induced primary esophageal squamous cell carcinoma protocol in mice, mouse esophageal tissue samples, and corresponding pathological stages. Scale bar: 30 μm.

[0142] 7. Three-dimensional visualization of blood vessels during the evolution of esophageal squamous cell carcinoma.

[0143] Three-dimensional fluorescence staining revealed that blood vessels gradually become disordered and tortuous during the development and progression of esophageal squamous cell carcinoma, such as... Figure 7 As shown in Figure (a). Furthermore, in invasive carcinoma, several large blood vessels were observed extending from the outer layer of muscle into the tissue mucosa, and radiating upwards from the base of the tumor mass into its interior, as shown in Figure (a). Figure 7 As shown by the arrow in (b). Figure 7 Three-dimensional images of esophageal vessels at different pathological stages are shown. (a) shows the three-dimensional projection (left column) and optical section (right column) of the vessels. The white dashed line in the right column depicts the epithelial margin. Arrows indicate large vessels extending from the muscular layer into the epithelium. Left column scale bar: 300 μm (NOR-DYS), 400 μm (ICA). Right column scale bar: 100 μm (NOR-DYS), 200 μm (ICA). (b) shows the bottom or side view of the radial vessels in three esophageal squamous cell carcinoma masses. Scale bar: 100 μm.

[0144] Further examination of the imaging depth showed that the fluorescence signal attenuation was less than 30% at a depth of 1000 μm. Figure 8 As shown in Figure (b), the image contrast remains high, as... Figure 8 As shown in Figure (a). These results demonstrate that images obtained using light-sheet microscopy can be well used for subsequent observation and analysis. Figure 8 The results show the imaging depth of light-sheet microscopy. (a) An optical section of a CD31-positive vessel (red) shows that the signal-to-noise ratio remains high at an imaging depth of 1000 μm. (b) Imaging depth detection of an esophageal tissue with a thickness >1200 μm.

[0145] 8. Vascular remodeling of the esophageal mucosa

[0146] Esophageal squamous cell carcinoma is a malignant tumor originating from the mucosal epithelium, and dysbiosis of the esophageal mucosal microenvironment is a key factor in its carcinogenesis and progression. In each CD31-stained three-dimensional vessel, three different cubical-shaped esophageal mucosal or tumor regions were randomly selected for computational modeling. Considering the different locations of the esophageal mucosa on different XY planes, the volume of each cube was approximately 700 × 250 × 40 μm. 3 Up to 600×300×60μm 3 ,like Figure 9 Figure (a) shows the vascular computational model. The vascular computational model was generated both automatically and manually to ensure accurate alignment. We analyzed the vascular structures in five pathological stages, as shown in Figure (a). Figure 9 Figure (b) shows the normal phase (NOR phase), pre-inflammatory phase (PINF phase), inflammatory phase (INF phase), dysplastic phase (DYS phase), and tumor area in invasive carcinoma (ICA). Figure 9 Computational modeling of blood vessels is shown in Figure (a). Figure (a) shows three randomly selected 3D cubic regions within each esophageal epithelium (three esophagus regions were analyzed at each pathological stage). Red: Blood vessels. Green: Computational model of blood vessels. Scale bar: 300 μm (whole esophagus), 20 μm (all analyzed regions). Figure (b) shows the original 3D images (above) and computational models (below) of blood vessels at five pathological stages. Scale bar: 20 μm.

[0147] Analysis shows that vascular remodeling, characterized by a decrease in vessel volume, length, area, and diameter, and an increase in vascular branching, occurs in the pre-inflammatory phase and is maintained from the pre-inflammatory phase to dysplasia; while almost the opposite structural changes are observed in invasive carcinoma, such as... Figure 10 Figures (a) to (e) are shown. Furthermore, during tumor progression, vascular perfusion resistance continuously decreases, such as... Figure 10 As shown in Figure (f). Figure 10This image shows vascular analysis of esophageal squamous cell carcinoma at different pathological stages; (a) vessel volume, (b) vessel length, (c) vessel area, (d) vessel diameter, (e) number of vessel branches, and (f) perfusion resistance. Perfusion resistance is directly proportional to vessel length and inversely proportional to vessel diameter, calculated using the formula ∑(lengthab / rarb). Pairwise comparisons were performed using a two-tailed unpaired t-test or the Mann-Whitney U test. Data are expressed as mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates no statistically significant difference.

[0148] It is noteworthy that the vascular ring structure is continuously increasing, such as Figure 11 As shown in Figures (a) and (b), the straightness of the blood vessels decreases, as... Figure 11 As shown in Figure (c), the vascular ring structure represents vascular connections, and its increase, along with the increase in vascular tortuosity (i.e., decreased vascular straightness), contributes to the redistribution of blood flow and the increase in tissue oxygen partial pressure. Figure 11 Figures show the changes in vascular ring structure and straightness; (a) 3D schematic diagram (red) and computational model (green) of vascular ring structure during the NOR and DYS periods. Scale bar: 20 μm. (b) Relative proportions of vascular rings (branch pts / terminal pts). (c) Quantitative analysis of vascular straightness. Pairwise comparisons between the NOR period and another period within the same group were performed using a two-tailed unpaired t-test or the Mann-Whitney U test. Data are expressed as mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates no statistical difference.

[0149] 9. Three-dimensional visualization and analysis of T cells and T cell aggregates during carcinogenesis.

[0150] Due to the positive interaction between blood vessels and T cells, three-dimensional visualization of T cells was further performed. To eliminate false positives, this study used a custom-designed 10× light sheet objective to perform three-dimensional imaging of CD3-stained T cells, such as... Figure 12 As shown in Figure (a). We performed a detailed evaluation of staining specificity to eliminate potential false positive results, such as Figure 12 As shown in Figure (b). CD3 infiltration in the esophagus. + T cells are located in the mucous membrane (e.g., ... Figure 12 (a) Right column of the figure) or muscle layer, but the latter has a significantly lower T cell density, such as Figure 12 Figure (c) shows the role of CD3 in the development and progression of esophageal squamous cell carcinoma. +T cells exhibit aggregation within the epithelium, forming characteristic T cell aggregates, such as... Figure 12 (d) diagram. Figure 12 This is a spatiotemporal dynamic distribution map of T cells and T cell aggregates. (a) CD3 + Three-dimensional projection of T cells (yellow) at five pathological stages (left column) and optical sections of the epithelial layer (right column). CD3 cells are circled in white. + T cell aggregates. Left column scale bar: 300 μm (NOR-DYS), 400 μm (ICA). Right column scale bar: 100 μm (NOR-DYS), 200 μm (ICA). (b) Magnified T cell image from light sheet microscopy. Scale bar: 15 μm. (c) Low T cell density in the muscle layer as shown in XY-axis optical sections. Scale bar: 200 μm. (d) Different XY-section views and three-dimensional projection of a T cell aggregate. Scale bar: 15 μm.

[0151] Quantitative analysis showed that epithelial CD3 + T cell density initially increases and then decreases, peaking during the inflammatory phase. Figure 13 As shown in Figure (a), the dynamic changes of T cell aggregates are consistent with those of T cells, but T cell aggregates are absent within the tumor, as... Figure 13 Figure (b) shows the distribution characteristics of T cells within the tumor, based on CD3. + The T cells were grouped based on their distance from the tumor margin. The results showed that T cell density was higher at the tumor margin and gradually decreased with increasing distance, exhibiting a structured spatial distribution. Figure 13 As shown in Figure (c). Figure 13 Figure 1 shows the results of quantitative T cell analysis. (a) Spatial density changes of T cells analyzed using Imaris software. (b) Density changes of T cell aggregates. No T cell aggregates were found inside the tumor during the ICA stage. * indicates comparison with the NOR group. *P<0.05; **P<0.01; ***P<0.001; ns, no statistical significance. (c) Three-dimensional projection (left), rendering (middle), and percentage of T cells at different locations along the tumor edge (right, n=3). In the rendering, the tumor edge is outlined by a white line, and T cells at different distances from the tumor edge are marked with dots of different colors. Scale bar: 100μm.

[0152] Tissue clearing technology has enabled three-dimensional labeling and imaging of whole-body and localized human organs in mice in recent years. With the increasing success of clearing methods in various tissues, the experimental challenges of tissue clearing technology have gradually shifted to the three-dimensional staining process, which is typically influenced by factors such as tissue characteristics, antibody selection, staining time, and procedure. Previously commonly used passive labeling methods (such as iDISCO and CUBIC) mainly rely on transgenic proteins, nanobodies, and small molecule dyes, while other IgG antibodies often struggle to stably penetrate various esophageal tissue layers within the recommended timeframe. While active labeling methods using electric cardiac pumps may be more effective, they are costly for individual tissues and require specific equipment and conditions, such as CLARITY and wildDISCO. Therefore, we optimized two cost-effective passive staining protocols and screened suitable antibodies for subsequent three-dimensional staining.

[0153] Histopathological staging of the original tissue is a crucial step in differentiating the stages of a lesion. Traditional spatial omics typically uses adjacent tissue sections to corroborate the pathological staging of experimental sections. Although not in situ verification, adjacent sections often exhibit similar pathological stages due to their thinness. However, in three-dimensional tissues, the pathological staging cannot be determined by adjacent sections, and due to the requirements of three-dimensional staining, pathological staging must be performed after clearing. In 2017, the DIPCO method was first reported as a pathological staining protocol for cleared tissues, but given the limited number of studies on this topic, this protocol remains only seen in a very small number of publications. Furthermore, considering the different tissue characteristics, we still need to optimize the DIPCO protocol to adapt to the precise pathological staging of mouse esophageal lesions. In previous experiments, we found that cleared mouse esophagus wrinkled after the routine dehydration and embedding steps, thus affecting the H&E staining effect. This may be due to the dehydration and defatting steps performed on the mouse esophagus and its small size. Therefore, we shortened the ethanol dehydration and xylene clearing time in paraffin embedding to reduce tissue wrinkling. In addition, we conducted professional histopathological examinations to ensure that this protocol can be used for pathological staging of transparent esophagus.

[0154] Although previous studies have confirmed significantly increased vascular density in esophageal squamous cell carcinoma (ESCC) tissue sections, the timing and specific changes in vascular remodeling within ESCC remained unclear. Our results show that vascular remodeling in mouse ESCC occurs as early as the pre-inflammatory stage and persists until the dysplastic stage, primarily manifested as decreased vessel diameter, volume, area, and length, while increasing vascular branching. Another interesting finding is the continuous increase in vascular ring structure and tortuosity before invasive carcinoma formation, which may indicate improved tissue oxygen partial pressure and blood flow redistribution. Vascular tortuosity is a common indicator in angiography and has been used in clinical screening. Furthermore, in the invasive stage of ESCC, the increased vessel diameter and tortuosity, along with reduced ring structure, suggest significant alterations in vascular structure that contribute to the formation of a relatively hypoxic microenvironment to attract and aggregate immune cells within the tumor.

[0155] Our quantitative in situ 3D analysis revealed the dynamic distribution characteristics of T cells and T cell aggregates during tumor progression. Previous studies have shown that T cells rely on functional blood vessels to enter tissues, and that there are positive regulatory interactions between them. Our results also validated the specific distribution of T cells near blood vessels in three-dimensional space. Furthermore, dynamic comparison with a three-dimensional random distribution model revealed that the distance between T cells and blood vessels may be related to T cell infiltration dynamics, with the shortest distance occurring during the inflammatory phase. Specifically, T cells tend to be distributed 8–12 μm from blood vessels under physiological conditions (NOR phase) and the potentially non-invasive phase (DYS phase), while in the inflammatory phase (INF phase), T cells are randomly distributed around blood vessels. In addition, T cells are more concentrated at a distance of 4–8 μm from blood vessels in the pre-inflammatory phase (PINF phase) and invasive carcinoma (ICA phase), a distance that may be related to the area of ​​immune cell infiltration. In addition to comparing the spatial distances, we further analyzed their functional interactions. However, given the indivisible nature of the vascular network, we focused our research on the interaction between T cell aggregates and blood vessels. We found that the T cell aggregate is CD3 + The aggregation of T cells initially increases and then decreases during tumor progression, peaking during the inflammatory phase. However, T cell aggregates are completely absent from the tumor core. These characteristics are highly similar to the phenotype of tertiary lymphoid structures. Tertiary lymphoid structures contain CD3+ cells... + T cells surround CD20 +B cell formation areas are organized aggregates of immune cells acquired in non-lymphoid tissues under non-physiological conditions, typically found in autoimmune diseases, chronic infections, and chronic inflammatory tissues caused by cancer. Infiltration of tertiary lymphoid structures is closely related to immune cell recruitment, favorable prognosis of immunotherapy, secretion of vascular cell adhesion molecule 1 (VCAM1), and angiogenesis. Based on in-depth analysis of the interaction between T cell aggregates and vascular networks, we found that T cell aggregates play a crucial role in driving vascular remodeling in esophageal squamous cell carcinoma, especially during the inflammatory phase. Specifically, we analyzed the characteristics of T cell aggregate-driven vascular remodeling, showing that it is particularly prominent during the inflammatory phase, mainly including changes in vessel length, area, tortuosity, and perfusion resistance, while other structural parameters remained largely unchanged. These results suggest the key role of chronic inflammation in angiogenesis in esophageal squamous cell carcinoma.

[0156] In summary, this study combined an optimized whole-tissue immunolabeling protocol with a DIPCO-based pathological staging scheme and utilized a custom-designed light-sheet microscope objective to achieve three-dimensional imaging and analysis of multi-stage lesions in mouse esophageal squamous cell carcinoma. The workflow established in this study provides new research methods and ideas for exploring tumor progression events, changes in the three-dimensional structure of immune cells or blood vessels in esophageal squamous cell carcinoma or other cancers.

[0157] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of this application.

Claims

1. A method for tissue transparency in the esophagus of mice at multiple stages of cancerous transformation, characterized in that: This includes whole-tissue immunostaining, clearing, three-dimensional imaging, histological staging, and image analysis procedures. The whole-tissue immunostaining step includes using screened antibodies to perform whole-tissue immunostaining on mouse esophageal samples; The transparency step includes making the immunolabeled mouse esophageal sample transparent; The three-dimensional imaging step includes performing three-dimensional imaging on a transparent mouse esophageal sample; The histological staging steps include de-transparentifying the mouse esophageal sample after three-dimensional imaging, paraffin embedding, sectioning, and pathological staining of the de-transparent mouse esophageal sample, and determining the pathological stage of the sample based on the pathological staining results. The image analysis step includes using analysis software to analyze three-dimensional images of tissues in the same pathological stage and to perform intergroup comparison analysis.

2. The tissue transparency method according to claim 1, characterized in that: The antibodies used in the whole-tissue immunostaining step are rat anti-CD31 and / or rabbit anti-CD3.

3. The tissue transparency method according to claim 1, characterized in that: The whole-tissue immunostaining step involves whole-tissue immunolabeling of mouse esophageal samples, specifically including one of the following two methods. 1) Passive Wild DISCO immunostaining protocol: The mouse esophageal sample to be stained is incubated in Wild DISCO permeation blocking solution, and then passively incubated in Wild DISCO immunostaining solution containing primary antibody. After incubation, it is washed with PBS. Then, the mouse esophageal sample is incubated in Wild DISCO immunostaining solution containing secondary antibody. After incubation, it is washed with PBS. This completes the staining of the entire mouse esophageal tissue. 2) iDISCO centrifugation staining protocol: Pretreated mouse esophageal samples to be stained were incubated in iDISCO permeation buffer and iDISCO blocking buffer, respectively. Then, the mouse esophageal samples were incubated in iDISCO primary antibody incubation buffer containing primary antibody. During incubation, the samples were centrifuged at 400-600g for 4-6 hours daily, while the antibody concentration of the primary antibody was gradually increased until the incubation in iDISCO primary antibody incubation buffer was completed. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature. Then, the mouse esophageal samples were incubated in iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI. Similarly, the samples were centrifuged at 400-600g for 4-6 hours daily. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature, thus completing the staining of the entire mouse esophageal tissue.

4. The tissue transparency method according to claim 3, characterized in that: In the passive Wild DISCO immunostaining protocol, the incubation time in 4-6 mL of Wild DISCO permeation blocking solution is 1-2 days, the incubation time in 1.5-2 mL of Wild DISCO immunostaining solution containing primary antibody is 7-10 days, and the incubation time in 1.5-2 mL of Wild DISCO immunostaining solution containing secondary antibody is 7-10 days. After primary antibody incubation, the washing method with PBS is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the washing method with PBS is 10-20 mL for 2-2.5 days, 10-12 times. Preferably, in the iDISCO centrifugal staining protocol, the incubation time in 4-6 mL of iDISCO permeation buffer is 1.5-2 days, the incubation time in 4-6 mL of iDISCO blocking buffer is 2-3 days, the incubation time in 1.5-2 mL of iDISCO primary antibody incubation buffer containing primary antibody is 3.5-4.5 days, and the incubation time in 1.5-2 mL of iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI is 3.5-4.5 days. After primary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 10-12 times.

5. The tissue transparency method according to any one of claims 1-4, characterized in that: The histological staging steps specifically include: immersing the transparent mouse esophageal samples in methanol aqueous solution with gradually decreasing concentrations, then washing them overnight in PBS to remove the transparency; then, gradually dehydrating the detransparent samples in ethanol aqueous solution with gradually increasing concentrations, treating them with xylene, and then impregnating them with paraffin.

6. The tissue transparency method according to claim 5, characterized in that: In the histological staging process, the methanol-water solution with gradually decreasing concentration is 100% methanol, 80% methanol-water solution, 60% methanol-water solution, 40% methanol-water solution, and 20% methanol-water solution. Preferably, the soaking method in the methanol aqueous solution with gradually decreasing concentration is as follows: first, soak in 100% methanol at least twice, each time using 4-6 mL of 100% methanol for 1-1.2 hours; then, soak in 4-6 mL of 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, and 20% methanol aqueous solution for 1-1.2 hours respectively. Preferably, after soaking in methanol aqueous solution, the sample is washed overnight in 15-50 mL of PBS; Preferably, the ethanol aqueous solution with gradually increasing concentration is 75% ethanol aqueous solution, 85% ethanol aqueous solution, 90% ethanol aqueous solution, 95% ethanol aqueous solution, and 100% ethanol; Preferably, the dehydration is carried out gradually in ethanol aqueous solutions with progressively increasing concentrations by soaking in 75% ethanol aqueous solution, 85% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution for 40-60 minutes respectively, and then soaking in 100% ethanol at least twice, each time for 30-40 minutes. Preferably, the xylene treatment includes immersion in xylene at least twice, each time for 20-30 minutes.

7. An immunostaining method for the esophagus of mice at multiple stages of cancerous transformation, characterized in that: This includes whole-tissue immunostaining of mouse esophageal samples using rat anti-CD31 and / or rabbit anti-CD3.

8. The immunostaining method according to claim 7, characterized in that: The whole-tissue immunostaining of mouse esophageal samples specifically includes one of the following two methods. 1) Passive Wild DISCO immunostaining protocol: The mouse esophageal sample to be stained is incubated in Wild DISCO permeation blocking solution, and then passively incubated in Wild DISCO immunostaining solution containing primary antibody. After incubation, it is washed with PBS. Then, the mouse esophageal sample is incubated in Wild DISCO immunostaining solution containing secondary antibody. After incubation, it is washed with PBS. This completes the staining of the entire mouse esophageal tissue. 2) iDISCO centrifugation staining protocol: Pretreated mouse esophageal samples to be stained were incubated in iDISCO permeation buffer and iDISCO blocking buffer, respectively. Then, the mouse esophageal samples were incubated in iDISCO primary antibody incubation buffer containing primary antibody. During incubation, the samples were centrifuged at 400-600g for 4-6 hours daily, while the antibody concentration of the primary antibody was gradually increased until the incubation in iDISCO primary antibody incubation buffer was completed. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature. Then, the mouse esophageal samples were incubated in iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI. Similarly, the samples were centrifuged at 400-600g for 4-6 hours daily. After incubation, the mouse esophageal samples were washed with PTwH solution at room temperature, thus completing the staining of the entire mouse esophageal tissue.

9. The immunostaining method according to claim 8, characterized in that: In the passive Wild DISCO immunostaining protocol, the incubation time in 4-6 mL of Wild DISCO permeation blocking solution is 1-2 days, the incubation time in 1.5-2 mL of Wild DISCO immunostaining solution containing primary antibody is 7-10 days, and the incubation time in 1.5-2 mL of Wild DISCO immunostaining solution containing secondary antibody is 7-10 days. After primary antibody incubation, the washing method with PBS is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the washing method with PBS is 10-20 mL for 2-2.5 days, 10-12 times. Preferably, in the iDISCO centrifugal staining protocol, the incubation time in 4-6 mL of iDISCO permeation buffer is 1.5-2 days, the incubation time in 4-6 mL of iDISCO blocking buffer is 2-3 days, the incubation time in 1.5-2 mL of iDISCO primary antibody incubation buffer containing primary antibody is 3.5-4.5 days, and the incubation time in 1.5-2 mL of iDISCO secondary antibody incubation buffer containing secondary antibody and DAPI is 3.5-4.5 days. After primary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 8-10 times. After secondary antibody incubation, the washing method with PTwH solution is 10-20 mL for 2-2.5 days, 10-12 times.

10. The application of an antibody in the preparation of a tissue clearing kit for mouse esophagus at multiple stages of cancer, characterized in that: The antibody is rat anti-CD31 and / or rabbit anti-CD3.

Images